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Advances in Experimental Medicine and Biology 1406

EimanAbdelMeguid

PritiL.Mishall

HaleyL.Nation

PaulM.ReaEditors

Biomedical

Visualisation

Volume 15 ‒ Visualisation in Teaching

ofBiomedical and Clinical Subjects: Anatomy,

Advanced Microscopy and Radiology

Advances in Experimental Medicine

and Biology

Volume 1406

Series Editors

Wim E. Crusio, Institut de Neurosciences Cognitives et Intégratives

d’Aquitaine, CNRS and University of Bordeaux, Pessac Cedex, France

Haidong Dong, Departments of Urology and Immunology, Mayo Clinic,

Rochester, MN, USA

Heinfried H. Radeke, Institute of Pharmacology and Toxicology, Clinic of the

Goethe University Frankfurt Main, Frankfurt am Main, Hessen, Germany

Nima Rezaei , Research Center for Immunodeﬁciencies, Children’s

Medical Center, Tehran University of Medical Sciences, Tehran, Iran

Ortrud Steinlein, Institute of Human Genetics, LMU University Hospital,

Munich, Germany

Junjie Xiao, Cardiac Regeneration and Ageing Lab, Institute of

Cardiovascular Sciences, School of Life Science, Shanghai University,

Shanghai, China

Advances in Experimental Medicine and Biology provides a platform for

scientiﬁc contributions in the main disciplines of the biomedicine and the

life sciences. This series publishes thematic volumes on contemporary

research in the areas of microbiology, immunology, neurosciences, biochem-

istry, biomedical engineering, genetics, physiology, and cancer research.

Covering emerging topics and techniques in basic and clinical science, it

brings together clinicians and researchers from various ﬁelds.

Advances in Experimental Medicine and Biology has been publishing

exceptional works in the ﬁeld for over 40 years, and is indexed in SCOPUS,

Medline (PubMed), EMBASE, BIOSIS, Reaxys, EMBiology, the Chemical

Abstracts Service (CAS), and Pathway Studio.

2021 Impact Factor: 3.650 (no longer indexed in SCIE as of 2022)

Eiman Abdel Meguid •Priti L. Mishall

Haley L. Nation •Paul M. Rea

Editors

Biomedical Visualisation

Volume 15 –Visualisation in

Teaching of Biomedical and Clinical

Subjects: Anatomy, Advanced

Microscopy and Radiology

Editors

Eiman Abdel Meguid

Queen’s University Belfast

Belfast, UK

Priti L. Mishall

Departments of Pathology &

Ophthalmology and Visual Sciences,

Albert Einstein College of Medicine

Bronx, NY, USA

Haley L. Nation

Department of Cell Systems

and Anatomy

UT Health Science Center

at San Antonio

San Antonio, TX, USA

Paul M. Rea

Anatomy Facility, School of Medicine,

Dentistry and Nursing, College of

Medical, Veterinary and Life Sciences

University of Glasgow

Glasgow, UK

ISSN 0065-2598 ISSN 2214-8019 (electronic)

Advances in Experimental Medicine and Biology

ISBN 978-3-031-26461-0 ISBN 978-3-031-26462-7 (eBook)

https://doi.org/10.1007/978-3-031-26462-7

#The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature

Switzerland AG 2023

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Preface

This book, the 15

volume of the Biomedical Visualization series, focuses on

scientiﬁc visualization. It encapsulates the integration of science and imaging.

This volume spans from the microarchitecture of speciﬁc proteins to recent

advancements in anatomy education, to the clinical application of new

technologies and the improvement of patient care. It is the intersection

between technology, science, history, creativity, and engineering. This book

demonstrates how we can teach and learn in a much more accessible, innova-

tive, and engaging way using technology.

This volume is particularly focused on three main themes: advanced

microscopy, anatomy education, and radiology visualization related to patient

care. The chapters pertaining to advanced microscopy convey complex bio-

medical information by visual means. These chapters provide an overview of

the principles of microscopy and the unique characteristics of different

microscopes; it also provides examples of applications of microscopy that

have led to groundbreaking novel discoveries. This volume also delves into

the concepts of education. These chapters summarize the recent trends in

anatomy education and emphasize the creation and use of existing online tools

to support student learning to understand complex data and abstract ideas.

Lastly, the radiological visualization segment dives into the history of radio-

graphic imaging, surgical visual instruments, and therapies in medicine and

dentistry. This volume highlights the profound effect technology has had on

improving patient outcomes and also foreshadows where future advancements

might take us.

This volume will be of particular interest to many; the scope of this

textbook encompasses medicine, dentistry, allied health professions, biomed-

ical sciences, anatomy education, radiology, and microscopy. Students and

researchers who have interests in microscopy and host–pathogen communi-

cation will ﬁnd these chapters useful in expanding their knowledge and

understanding microscopies application. Gross anatomy and histology

instructors will clearly enjoy the advancements in education explored in

these chapters. However, the educational topics discussed are applicable to

all higher education staff and across all basic science disciplines such as health

care practitioners, ranging from dentists to orthopedists. This volume

highlights the clinical importance of advancements in visualization pertaining

to patient care. It is our hope that students, educators, researchers, and

clinicians reading this book will learn something new, are stimulated to ask

innovative questions, and are inspired to continue the technological

advancements pushing science forward.

vi Preface

Belfast, UK Eiman Abdel Meguid

Bronx, NY Priti L. Mishall

San Antonio, TX Haley L. Nation

Glasgow, UK Paul M. Rea

Contents

Part I Microscopy

1 Advances in Microscopy and Its Applications with Special

Reference to Fluorescence Microscope: An Overview ....... 3

N. B. Pushpa, Apurba Patra, and Kumar Satish Ravi

2 Visualisation of Host–Pathogen Communication .......... 19

Amy Dumigan, Ricardo Calderon Gonzalez, Brenda Morris,

and Joana Sá-Pessoa

3 A Review of Pathology and Analysis of Approaches to Easing

Kidney Disease Impact: Host–Pathogen Communication

and Biomedical Visualization Perspective ................ 41

Kacper Pizon, Savita Hampal, Kamila Orzechowska,

and Shahid Nazir Muhammad

Part II Radiology and Patient Care

4 The Evolution of Equipment and Technology for

Visualising the Larynx and Airway .................... 61

Duncan King and Alison Blair

5 The Impact of Technological Innovation on Dentistry ...... 79

Richard Zimmermann and Stefanie Seitz

6 Advanced 3D Visualization and 3D Printing in Radiology . . . 103

Shabnam Fidvi, Justin Holder, Hong Li, Gregory J. Parnes,

Stephanie B. Shamir, and Nicole Wake

7 3D Visualisation of the Spine ......................... 139

Scarlett O’Brien and Nagy Darwish

Part III Anatomy Education

8 Visualization in Anatomy Education ................... 171

Apurba Patra, Nagavalli Basavanna Pushpa,

and Kumar Satish Ravi

vii

viii Contents

9 Visualizing Anatomy in Dental Morphology Education ..... 187

Tamara Vagg, Andre Toulouse, Conor O’Mahony,

and Mutahira Lone

10 Flashcards: The Preferred Online Game-Based Study

Tool Self-Selected by Students to Review Medical

Histology Image Content ............................ 209

Priti L. Mishall, William Burton, and Michael Risley

About the Editors

Eiman Abdel Meguid PhD, MBBS, SFHE, is Fellow of the Anatomical

Society. She is a medical graduate and Senior Lecturer of Anatomy at Queen’s

University Belfast (QUB), UK, with expertise in human anatomy, embryol-

ogy, and neuroanatomy. She is passionate about anatomical sciences educa-

tion and is keen to actively support the education of her students. Eiman’s

areas of professional interest include anatomical variation, multimodal teach-

ing strategies, implementing innovative technology, audience response sys-

tem in teaching, ﬂipped classrooms, and blended learning. She has published

widely and presented at many international meetings that included invited

talks. She conducts review sessions for clinical trainee revisiting the cadaveric

anatomy labs whenever required. She is the Sole Academic Lead for Dental

Anatomy Course, Co-lead for Musculoskeletal Unit 2 for the MB BCh BAO,

and MSc in Clinical Anatomy Programme. She has multiple publications in

high impact journals and was an International Visiting Scholar/Guest Speaker

for Weill Cornell Medicine, NY. Dr. Abdel Meguid is the recipient of the

Senior Faculty Award for best presentation at the AACA conference, 2022.

She has previously acted as a member of the Career Development Committee

of the American Association of Clinical Anatomy (AACA), and currently, she

is a member of the Educational Affairs Committee of AACA. She is a

reviewer for the Clinical Anatomy, BMC Medical Education, and the

Anatomical Sciences Education Journals. Through her membership of the

Risk Management Group Committee at the Royal College of Surgeons and

previous role at the Research Ethics Committee at QUB, she gained lot of

experience in reviewing and generating policies for educational and research-

related activities. She is involved in peer mentoring as she is passionate about

Staff Professional Career Development. Having spent many years teaching as

a clinical anatomist, she is well placed to assist in producing resources that

would assist the development of student education. The extensive experience

she has gained, her international networking, and her interest in tailoring

teaching and research have equipped her with the necessary skills.

Priti L. Mishall MD, MBBS, PGCertME, is Associate Professor in the

Departments of Pathology & Ophthalmology and Visual Sciences. She is a

medical educator with expertise in human anatomy, embryology, histology,

and neuroanatomy. She teaches anatomy to learners in Undergraduate

Medical Education (UME) and Graduate Medical Education (GME). For the

UME, she Co-Directs the Anatomy course for the ﬁrst-year MD program and

also Directs the Anatomy course for the MSTP program. She is the Director of

the Anatomical Donation Program and is a course faculty member for the

second-year Nervous System and Human Behavior course. She is a small

group facilitator for the Problem-Based Learning sessions in Pre-clerkship

and Transition to Clerkship. Dr. Mishall mentors medical students interested

in pursuing projects on creating and evaluating anatomy education learning

resources. In the area of GME, she conducts workshops for residents and

fellows revisiting cadaver anatomy labs. Her clinical collaborators include

Departments of Ophthalmology and Visual Sciences and Rheumatology. She

is passionate about participating in faculty development initiatives and is a

course faculty at Harvard Macy Institutes’course “Transforming your teach-

ing for the Virtual Environment.”She is an active member of the American

Association of Clinical Anatomists (AACA) and International Association of

Medical Science Educators (IAMSE). She served as the chair of the Education

Affairs Committee of AACA and is the member of AACA’s Career Develop-

ment Committee. At Einstein, she implemented various e-learning instruc-

tional methods including ﬂipped classrooms, blended and online learning to

promote learner interactivity and engagement with the content. Additionally

she has expertise in designing and implementing prosection-based anatomy

course for preclerkship curriculas. She is a recipient of two teaching awards at

Einstein: the Samuel M. Rosen Award for Outstanding Teaching

(Pre-Clerkship) and the Harry Eagle Award for Outstanding Teaching

(Pre-Clerkship) in recognition for excellence in teaching by students and by

peers, respectively.

x About the Editors

Haley L. Nation PhD, is Associate Professor in the Department of Cell

Systems and Anatomy at UT Health San Antonio. She is a classically trained

anatomist who teaches various basic science disciplines to future health care

professionals, including gross anatomy, histology, neuroanatomy, and embry-

ology. Dr. Nation teaches medical, dental, physical therapy, physician assis-

tant, occupational therapy, and master’s of anatomy students. She is

committed to providing a high quality of education to her students as well

as various residents and practicing dentists seeking continuing education. She

is invested in the success of the programs she teaches in and the wider

academic missions as a whole; she serves on admissions, curriculum, and

strategic planning committees and partakes in leadership programs. To help

serve the greater anatomical community, she is involved in professional

associations and serves on the American Association of Clinical Anatomists

(AACA) Educational Affairs Committee (EAC) and Committee on Diversity,

Equity, and Inclusion (CDEI). In addition to her educational and service

commitments, she also has a particular interest in mentoring graduate students

in research projects involving anatomical variations and the creation of new

educational tools. Throughout her career she has been devoted to her students,

institution, and anatomical community.

About the Editors xi

Paul M. Rea Paul is Professor of Digital and Anatomical Education at the

University of Glasgow. He is Director of Innovation, Engagement and Enter-

prise within the School of Medicine, Dentistry and Nursing. He is also a

Senate Assessor for Student Conduct and Council Member on Senate and

coordinates the day-to-day running of the Body Donor Program and is a

Licensed Teacher of Anatomy, licensed by the Scottish Parliament. He is

qualiﬁed with a medical degree (MBChB), an MSc (by research) in craniofa-

cial anatomy/surgery, a PhD in neuroscience, the Diploma in Forensic Medi-

cal Science (DipFMS), and an MEd with Merit (Learning and Teaching in

Higher Education). He is Senior Fellow of the Higher Education Academy and

a Fellow of the Institute of Medical Illustrators (FIMI). Paul has published

widely and presented at many national and international meetings, including

invited talks. He has been the lead editor for Biomedical Visualisation over

13 published volumes and is the founding editor for this book series. This has

resulted in almost 90,000 downloads across these volumes, with approxi-

mately 400 different authors, across approximately 100 institutions from

19 countries across the globe. He is Associate Editor for the European Journal

of Anatomy and has reviewed for 25 different journals/publishers. He is the

Public Engagement and Outreach lead for anatomy coordinating collaborative

projects with the Glasgow Science Centre, NHS, and Royal College of

Physicians and Surgeons of Glasgow. Paul is also a STEM ambassador and

has visited numerous schools to undertake outreach work.

His research involves a long-standing strategic partnership with the School of

Simulation and Visualisation, The Glasgow School of Art. This has led to

multi-million-pound investment in creating world-leading 3D digital datasets

to be used in undergraduate and postgraduate teaching to enhance learning

and assessment. This successful collaboration resulted in the creation of the

world’sﬁrst taught MSc Medical Visualisation and Human Anatomy combin-

ing anatomy and digital technologies. The Institute of Medical Illustrators also

accredits it. It has created college-wide, industry, multi-institutional, and NHS

research linked projects for students.

Part I

Microscopy

Advances in Microscopy and Its

Applications with Special Reference

to Fluorescence Microscope:

An Overview

N. B. Pushpa, Apurba Patra, and Kumar Satish Ravi

Abstract

The microscope has revolutionized the under-

standing of an organism’s structural details

and cellular functions. With the invention of

highly evolved microscopes, the diagnosis and

treatment of diseases has gained momentum.

Technology has immensely helped demon-

strate cellular events like phagocytosis, cell

movement, cell division, etc. with enhanced

temporal and spatial resolution. One of these

advanced inventions is the ﬂuorescent micro-

scope which has enabled scanning through

various physiological activities of the cell. A

ﬂuorescence microscope uses the property of

ﬂuorescence to create an image. In addition to

visualizing the structural details of the cells, a

ﬂuorescence microscope also aids in

witnessing cellular activities. With an immu-

noﬂuorescence microscope, cellular antigens

can be localized. This chapter highlights the

basics of microscopy, types of microscopes,

principles, and types of ﬂuorescence

microscopes, and recent advances in micros-

copy and its application.

N. B. Pushpa

Department of Anatomy, JSS Medical College,

JSSAHER, Mysore, Karnataka, India

A. Patra

Department of Anatomy, All India Institute of Medical

Sciences, Bathinda, Punjab, India

K. S. Ravi (✉)

Department of Anatomy, All India Institute of Medical

Sciences, Rishikesh, Uttarakhand, India

#The Author(s), under exclusive license to Springer Nature Switzerland AG 2023

E. Abdel Meguid et al. (eds.), Biomedical Visualisation, Advances in Experimental Medicine and

Biology 1406, https://doi.org/10.1007/978-3-031-26462-7_1

Keywords

Microscopy · Fluorescence · Inventions ·

Fluorescent antibody technique · Cell

division · Cell movement · Phagocytosis

1.1 Introduction

With the recent advances in science, an excellent

thirst for looking into the ultrastructure of

particles and organisms has emerged. This

paved the way for the evolution of a branch of

science—“Microscopy,”which refers to the

observation of minute details of the structure

that the naked eye cannot see. Recent

microscopes have assured the visualization of

particles from nanometers to millimeters in size.

The most common type of microscope univer-

sally used is the optical microscope. This micro-

scope produces an enlarged image of the sample

placed in the focal plane of the lens. The objective

piece has a real, magniﬁed picture of the sample

set at a deﬁned distance from its front lens. In

contrast, the objective of inﬁnite tube-length

microscopes is to produce a virtual image of the

sample at inﬁnity. Microscopy techniques have

revolutionized the ﬁeld of medicine by giving rise

to sub-sepcialties like histology and pathology.

Advanced research has been empowering

super-resolution techniques by enhancing magni-

ﬁcation, imaging mode, numerical aperture, spa-

tial resolution, and light wavelength (Abramowitz

and Davidson 2007). The older microscopes had

limitations in terms of resolution. Depending on

the need to visualize different structures and

organisms, different types of microscopes have

been invented. The invention of sophisticated

microscopes has resulted in a better understand-

ing of not only the structural details of the cell but

also the physiological activities (The University

of Edinburgh 2018).

4 N. B. Pushpa et al.

1.1.1 Magnification

In optics, magniﬁcation (×) refers to the size of an

image produced relative to the object’s size.

Linear magniﬁcation refers to the ratio

between the image length and the object length

measured at the right angle to the optical axis.

The term longitudinal magniﬁcation is used when

the image is enlarged along the optical axis. The

image is called inverted when the values of linear

magniﬁcation are negative. However, angular

magniﬁcation refers to the ratio of the tangents

of the angles formed by an object and its image

when measured from a given point in the micro-

scope, i.e., lens or magniﬁers. Theoretically, mag-

niﬁcation can be inﬁnite, but practically,

magniﬁcation caused by any optical instrument

is limited by its resolving power (Ray 2002;

Wayne 2021).

1.1.2 Resolving Power

Resolving power refers to the ability of a micro-

scope to produce precise, distinct images of very

closely placed objects. The objective lens’s

numerical aperture depicts the microscope’s abil-

ity to delineate the specimen at a ﬁxed distance.

The greater the distance between the two points,

the greater the resolution will be. The resolution

also depends on the property of the light used in

the microscope. Hence, if the light of a shorter

wavelength is used, then the resolution caused

will be more compared to the light of a greater

wavelength (Jonkman et al. 2003).

1.1.3 Parts of Light Microscope

(Fig. 1.1)

The essential parts of an optical microscope

include stage, slide holder, eyepiece, objective,

nosepiece, base, condenser, diaphragm, light

source, coarse and ﬁne adjustment, and carrying

handle. Objectives and condenser need a special

mention also. The objective piece of a microscope

can have a single or combination of lenses that

face the object and collects light from it. The

principal property of objective lenses is magniﬁ-

cation. The overall magniﬁcation provided will be

combined magniﬁcation with the eyepiece. For

example, an objective with 4×will provide

40 times magniﬁcation with a 10×eyepiece.

Three or four objectives of different

magniﬁcations are ﬁtted into one nosepiece,

which can rotate based on the required lens.

These objective lenses are color coded for easy

selection. Different magniﬁcations available are

4×,10×,40×, and 100×. The lowest magniﬁca-

tion is 4×, called an objective scanning lens,

Fig. 1.1 Schematic diagram of Compound microscope

showing its parts

while the highest is 100×, called the large objec-

tive lens.

1 Advances in Microscopy and Its Applications with Special Reference to... 5

The sample is illuminated by gathering light

from the source through condenser lenses that are

placed over the light source; resulting in a clear,

sharp image with optimal magniﬁcation. The

amount of light entering the condenser can be

controlled by the diaphragm. It is suggested to

use a condenser for higher magniﬁcation rather

than for low magniﬁcation. Condensers are

mainly of three types- chromatic (Abbe), apla-

natic, and compound achromatic condensers.

The aplanatic condensers are used to correct

spherical aberration, while compound achromatic

condensers are preferred to rectify both chromatic

and spherical aberrations. Although Abbe

condensers can be used for magniﬁcation of less

than 400×only, it is still the most widely used

microscope condenser. There are specialized

condensers for Phase contrast, dark ﬁeld, and

epiﬂuorescence microscopes. In epiﬂuorescence

microscopy, the objective lens functions as both a

magniﬁer and a condenser (Abbe 1874).

1.2 Evolution of Microscope

The idea of constructing a microscope dates back

to the thirteenth century and originated with a

hand-held lens to magnify objects (Atti 1975).

Hence, a simple lens with required magniﬁcation,

object stand, and adjustment apparatus

constituted a light microscope. This was followed

by the invention of the primitive form of the

compound microscope with two lenses by Dutch

eyeglass maker Zacharias Jansen in 1595, which

could magnify the specimen by nine times (Van

Helden et al. 2010; William 1996). Anton van

Leeuwenhoek invented a high magniﬁcation

microscope, later termed the simple microscope,

hence he is referred to as the “Father of Micros-

copy.”The construction of the compound micro-

scope followed this invention. Robert Hooke

further embellished the instrument with a stage,

light source, and controls for coarse and ﬁne

adjustment (Lane 2015). Robert Hooke was the

ﬁrst to use the term “cell”based on their appear-

ance under the microscope. Until the eighteenth

century, microscopes could only magnify up to

50×. Then Carl Zeiss and Ernst Abbe added a

substage condenser and improved the lenses,

which could provide better magniﬁcation and

resolution (Abbe 1874).

1.3 Principle of Microscopy

The objective piece of the microscope carries an

objective lens that focuses on the object and

forms an enlarged image of the object, referred

to as a primary image (I1). The resulting magniﬁ-

cation is known as primary magniﬁcation. This

primary image is a magniﬁed, inverted real

image, which acts as an object for the eyepiece

and is projected up into the focal plane of the

eyepiece (Inoué 2006). The eyepiece magniﬁes

the primary image and secondary image (I2) is

formed at about 10 inches from it (Fig. 1.2).

1.4 Types

Microscopes are categorized chieﬂy based on

how they interact with the specimen/sample to

produce the image. Depending upon the kind of

light source used, we have different types of

microscopes.

1.4.1 Optical Microscopy

1.4.1.1 Bright Field Microscopy

The basic form of a light microscope. The speci-

men under observation is illuminated by the light

source below and observed from above by the

eyepiece. The most signiﬁcant advantage is its

simple technology and easy preparation of the

slide used for the study. However, the major

drawback is its poor contrast of the specimen

and low apparent resolution (Chandler and

Roberson 2009).

1.4.1.2 Dark-Field Microscopy

This technique is preferred for ameliorating the

contrast of transparent specimens, which are

unstained (Abramowitz and Davidson 2007).

Darkﬁeld illumination utilizes an aligned light

source to limit the amount of transmitted light

passing through the image plane. This technique

functions by illuminating the structures under

observation with intense oblique peripheral

light. The central light beam is excluded from

approaching the objective lens by the central

stopper patch attached to the condenser. Its appli-

cation is appreciated in the study of extremely

tiny and transparent particles. Dark ﬁelds can

exponentially upgrade the image contrast—par-

ticularly of transparent objects. Although this

technique requires minimum setup or sample

preparation, the limitations of this technique are

low apparent resolution and diminished intensity

of the ﬁnal image (Mualla et al. 2018).

6 N. B. Pushpa et al.

Fig. 1.2 Principle of microscopy showing primary image and secondary image

1.4.1.3 Oblique Illumination

Microscope

This microscope gives a 3D appearance with the

use of oblique illumination and also highlights

invisible features. The main limitation is the low

contrast of various biological samples; the out-of-

focus object may result in low apparent resolu-

tion. Hoffmann’s modulation contrast is a recent

advancement in this technique and is used in

inverted microscopes for usage in cell culture.

The prime limitation in the oblique illumination

microscope is identical to those found in bright

ﬁeld microscopy (Chandler and Roberson 2009).

1.4.1.4 Phase Contrast Microscope

This works on the principle that the difference in

refractive indices of the media and the object

results in phase difference. Hence, an image will

be formed because of interference between direct

and diffracted light from the object. The greater

the phase difference the greater will be the differ-

ence in amplitude, and the brighter will be the

resulting image. Because of this principle, living

cells can be directly observed, hence can visualize

the cellular activities under a phase contrast

microscope without sacriﬁcing/staining proce-

dure. This enables us to understand ongoing

dynamic cellular events in their natural state

(Mualla et al. 2018).

1.4.1.5 Interference Microscope

This works on the principle of interference, where

a beam of light from the source is split (Mualla

et al. 2018).

1.4.1.6 Dispersion Staining Microscopy

It is a set of staining techniques utilized in

establishing the identity of an unknown minute

particle with the aid of standard particles of

known properties. The property utilized here is

the “dispersion curve”based on the refractive

index of the two particles. The difference in the

dispersion curve becomes apparent when they

intersect. This technique is mainly employed in

detecting asbestos in construction materials. The

novelty of this technique is that it is free from

dyes/coloring substances (Bruni et al. 1998).

1 Advances in Microscopy and Its Applications with Special Reference to... 7

1.4.1.7 Polarization Microscope

A conventional microscope with a rotating stage

has two polarizing elements and balsam. The

polarizer is ﬁtted below the condenser, and the

analyzer is attached above the objective lens. This

microscope is mainly utilized to differentiate

between amorphous and crystalline biological

specimens (Oldenbourg 2013).

1.4.1.8 Atomic Force Microscope

Offers very high resolution in terms of nanometer

fractions, a thousand times more than the regular

optical microscope. Here information is obtained

by touching/feeling the structure’s surface with a

mechanical probe (cantilever tip). Currently, this

is the only microscope that can provide structural,

functional, and mechanical information about the

cell at high resolution (Trache and Meininger

2008).

1.4.1.9 Fluorescence Microscope

Tissues are subjected to a ﬂuorescent dye, and

ultraviolet light falls on the object. The tissue

emits more long-wavelength light and becomes

visible against the dark background. Compared to

a traditional ﬂuorescence microscope, photon

microscopy provides ﬁne resolution of particles/

tissue less than 1 mm (Hama et al. 2011).

1.4.1.10 Confocal Microscopy

It uses a focused laser beam that is scanned across

the sample to excite ﬂuorescence point by point.

To prevent unfocused light from reaching the

detector, the emitted light is guided through a

pinhole, usually a photomultiplier tube. The

image is then compiled in a computer based on

the measured ﬂuorescence intensities and the

position of the excitation laser. Confocal micros-

copy provides slightly higher lateral resolution

and greatly improves axial resolution/optical

sections. Therefore, such microscopy is widely

used where the 3D structure of the sample is

essential (Pawley 2006).

1.4.2 Electron Microscopy

Here, electrons are used instead of light, and the

image is focused on the ﬂuorescent screen by

magnetic coils. The resolution provided by the

electron microscope is 3–5 angstroms; even the

ultrastructure of the cell/object can be visualized.

Since it offers a higher resolution, more subtle

details of the structure can be studied. In a trans-

mission electron microscope, the images are

formed due to the passage of electrons into the

object. In a scanning electron microscope, images

are formed based on the ability of the object to

emit secondary electrons from its surface. The

compound microscope had the limitation of

lower resolution due to the limited wavelength

of light used. This limitation is overcome by

electron microscopes, where an electron beam

with a much smaller wavelength is used to obtain

a higher resolution. A magnetic ﬁeld is used to

obtain electron-optical systems that function

much like the glass lenses of a compound micro-

scope. Electron microscopes are basically used to

observe and examine ultrastructure. They use

specialized digital cameras to create electron

micrographs to capture the image (Mualla et al.

2018).

Transmission electron microscopy (TEM) is a

traditional form of an electron microscope. In

this, a high-voltage electron beam is used to illu-

minate a very thin slice of the sample/specimen to

form an image. The source of the electron beam is

usually an electron nozzle with a tungsten ﬁla-

ment. The emerging electron beam through the

specimen acquires information about the

observed specimen and is further magniﬁed by

the objective lens. A serial section electron micro-

scope is used to obtain consecutive serial sections

of a particular structure. These sections of the 3D

volume are exposed to the electron beam, and a

complete image of the structure is then obtained

(Chandler and Roberson 2009).

Scanning Transmission Electron Microscopy

(STEM)—This transmission electron microscope

is equipped with a scanning capability. STEM

visualizes the details of a structure by detecting

electrons scattered through the structure. So, it

can provide a very clear 3D view with high

resolution.

8 N. B. Pushpa et al.

Scanning Electron Microscope (SEM)—It

uses a scanning probe and a focused electron

beam. SEM creates images reﬂecting their com-

position and topography. Because a certain

amount of energy is lost when the electrons inter-

act with the sample, the image produced in this

microscope has a slightly lower resolution com-

pared to TEM. In a reﬂection electron micro-

scope, the reﬂected electron beam is detected

instead of the secondary/transmitted electrons.

This principle is often combined with reﬂection

high energy diffraction (RHEED)/reﬂection high

energy loss spectroscopy (RHELS). Electron

microscopes are designed for X-ray spectroscopy

and can provide quantitative and qualitative ele-

mental analysis, also called analytical electron

microscopes, and are an effective tool for

analyzing nanomaterials (Kosasih and Ducati

2018).

1.4.3 Scanning Probe Microscopy

Aﬁxed probe is available in the microscope, the

tip of which makes physical contact with the

surface of the object. The atomic force micro-

scope (AFM), photonic force microscope, scan-

ning tunneling microscope, and repetition

monitoring microscope are various examples of

scanning probe microscopes (Mualla et al. 2018).

1.4.4 Ultrasonic Force Microscopy

(UFM)

This microscopic method was introduced to

improve the image contrast and detail on “ﬂat”

regions under study, where AFM images have

contrast limitations. AFM-UFM fusion enables

near-ﬁeld acoustic microscopic image generation.

The AFM tip enables the detection of ultrasonic

waves and overcomes the wavelength limitation

encountered in acoustic microscopy. The elastic

changes that occur under the AFM tip help create

an image with more detail than that produced by

AFM. In UFM, local elasticity mapping is

enabled in AFM by applying ultrasonic vibrations

to a cantilever or sample. In order to quantita-

tively analyze the UFM results, a force-distance

curve measurement is performed with ultrasonic

vibration applied to the cantilever base. Then the

results are compared with a dynamic model of the

cantilever and the tip-sample interaction

depending on the ﬁnite difference technique

(Dinelli et al. 2011).

1.4.5 Ultraviolet Microscopy

In this type, ultraviolet light is used as a source of

light. The amount of light absorbed by the object

is recorded photographically, and the molecules

in the object, like purine, pyrimidine, DNA, etc.,

are identiﬁed. This technique serves two main

purposes. The ﬁrst is the use of shorter wave-

length ultraviolet electromagnetic energy to

improve image resolution beyond standard opti-

cal microscopes. The second application is the

increase of contrast when the response of individ-

ual samples relative to their surroundings is

increased due to the interaction of light with the

molecules in the examined sample. One such

example is the contrast enhancement of protein

crystals that form in salt solutions (Heimann and

Urstadt 1990).

1.4.6 Infrared Microscopy

This type of microscopy refers to using infrared

waves in the microscope. In a typical microscope

conﬁguration, a Fourier transforms

infrared (FTIR) spectrometer is combined with

an infrared detector and an optical microscope.

The infrared detector can be a linear array/2D

focal plane array/single point detector. FTIR

enables chemical analysis using infrared spectros-

copy, then the microscope and point/matrix detec-

tor further allow this chemical analysis to be

spatially resolved, i.e., performed in different

areas of the sample. The technique itself is also

called infrared microspectroscopy. An alternative

arrangement known as laser direct infrared imag-

ing involves a combination of a tuneable infrared

light source, and a single-point detector mounted

on a ﬂying objective. This technique is often used

for infrared chemical imaging, where image con-

trast is due to the response of individual sample

regions to speciﬁc user-selected IR wavelengths,

speciﬁc IR absorption bands, and associated

molecular resonances. A prime limitation of con-

ventional infrared microspectroscopy is that spa-

tial resolution is limited by diffraction (Pollock

and Kazarian 2014).

1 Advances in Microscopy and Its Applications with Special Reference to... 9

1.4.7 Photoacoustic Microscopy

This microscopic technique relies on

the photoacoustic effect (Bell 1880), which is

the generation of ultra-level sound caused by the

absorption of light. A focused intensity

modulated laser beam is raster scanned across

the sample. The generated (ultra) sound is

detected using an ultrasonic transducer. Piezo-

electric ultrasonic transducers are commonly

used in photoacoustic microscopy to analyze the

structure (Yao and Wang 2013).

1.4.8 Digital Holographic Microscopy

In DHM, interfering wavefronts arising from a

coherent or monochromatic light source are

recorded on the sensor. The image created in

this way from the recorded hologram is digitally

reconstructed by a computer. In addition to the

normal image in the bright ﬁeld, an image with a

phase shift is also created. This technique can

control both transmission mode and reﬂection.

In transmission mode, the phase-shift image

enables a label-free quantitative measurement of

the optical thickness of the sample. While in

reﬂection mode, the phase shift image provides

a relative distance measurement and thus

represents a topographical map of the reﬂecting

surface. Phase shift images of biological cells are

very similar to stained cells and have been suc-

cessfully analyzed by high content analysis soft-

ware. A unique feature of DHM is its ability to

adjust focus even after the ﬁnal image is recorded,

as all focus planes are recorded simultaneously by

the hologram. This feature makes it easier to

capture moving objects. Another attractive fea-

ture is the DHM’s ability to use low-cost optics

with software-based optical aberration correction

(Rappaz et al. 2014).

1.4.9 Digital (Virtual Microscopy)

Digital pathology is made possible partly with

virtual microscopy, in which the glass slides are

converted into digital slides that can be viewed

and analyzed. This technique is a visual informa-

tion environment that enables a computer-aided

mechanism to manage information generated

from a digital slide (Mualla et al. 2018).

1.4.10 Laser Microscopy

In laser microscopy, the laser beam is utilized as

an illumination source (Duarte 2016). For exam-

ple, laser microscopy focused on biological

applications uses ultrashort pulse lasers in a vari-

ety of techniques referred to as saturation micros-

copy, nonlinear microscopy, and two-photon

excitation microscopy (Thomas and Rudolph

2008).

High-intensity, short-pulse laboratory X-ray

lasers are under evaluation. If this technology is

successfully developed, it will be possible to

obtain enlarged 3D images of basic biological

structures in vivo at a particular time. The resolu-

tion is limited primarily by the hydrodynamic

expansion that occurs when the required number

of photons is registered (Solem 1983). Thus, even

if a specimen is destroyed by exposure, its con-

ﬁguration may be captured before it explodes

(Solem 1982).

1.5 Fluorescence Microscopy

1.5.1 Principle and Mechanism

Aﬂuorescence microscope uses the property of

ﬂuorescence with other properties like reﬂection,

scattering, and absorption to produce the image.

Fluorescence is the emission of light by a sub-

stance that has absorbed light or electromagnetic

radiation (Gill 2010). The objects become visible

when absorbed radiation is ultraviolet (UV) rays

of the electromagnetic spectrum, and the struc-

ture/specimen under study emits visible light; this

provides the structure with distinct color to be

observed in the presence of UV light. Spectral

emission ﬁlters are used to separate the

illuminating light from emitted ﬂuorescence.

The ﬂuorescent structure stops glowing when

the radiation source is stopped (Fig. 1.3).

10 N. B. Pushpa et al.

Fluorescence occurs when a particle/atom

relaxes from a high to a lower energy state by

emitting a photon without altering the electron

spin. There is a loss of some energy in this pro-

cess resulting in photons with less power

(Helmchen and Denk 2005). Higher energy rays

have a short wavelength, and those with low

energy have a long wavelength; as a rule, the

emitted rays will have a longer wavelength called

Stokes shift. It is also possible for a source to

absorb energy at a time and emit a single photon

with less energy. This energy difference will

assign a different color to the particle than in

reality. Hence red light can be utilized to produce

green light. This is better achieved with photons

having high spatial and temporal density. That is

why red light is more preferred as an excitation

source over blue light since red light has more

penetration to the tissue because of its less

scattered nature. Also, the disadvantage of cell

damage with high-energy light can be overcome

with red light as an excitation source

(Zinselmeyer et al. 2009). Image brightness in a

ﬂuorescence microscope is directly proportional

to the number of photons gathered by the detector

during the speciﬁc dwell time. Other super-

resolution techniques like stimulated emission

depletion can overcome the default diffraction

limit and produce an image of signiﬁcant

magniﬁcation.

In the two-photon ﬂuorescence technique, two

photons are simultaneously excited, unlike in the

regular ﬂuorescence microscope. Here the

wavelength of the utilized light is more than that

of emitted light, in contrast to the single photon

technique. Often the excitation light is

near-infrared rays which can minimize the scat-

tering within the tissue. As two photons are

absorbed with each excitation, this technique

offers excellent resolution. Hence this is preferred

over confocal microscopy with added advantages

of decreased photobleaching, deep tissue penetra-

tion, clear background, and efﬁcient light detec-

tion (Denk et al. 1990).

1.5.2 Epifluorescence Microscopy

Most ﬂuorescence microscopes, particularly

those used in the life sciences, have an

epiﬂuorescence design, as shown in Fig. 1.3.

Light of the excitation-speciﬁc wavelength

illuminates the sample through an objective lens.

The ﬂuorescence emitted by the specimen follows

and is focused on the detector, which will need an

objective with a higher numerical aperture for

greater resolution. Since most of the excitation

light passes through the spsecimen, only the

reﬂected excitation light, together with the dis-

charge light, reaches the objective, thus providing

a high signal-to-noise ratio. The dichroic beam

splitter acts as a wavelength-speciﬁcﬁlter,

allowing only ﬂuorescent light into the eyepiece

or detector, but reﬂecting back any remaining

excitation light toward the source (Sanderson

et al. 2014).

1.5.2.1 Light Sources for Fluorescence

Fluorescence microscopy needs intense near

monochromatic illumination, which some

extended light sources, such as halogen lamps,

cannot provide (Huang 2010). Four types of light

sources are commonly used, including xenon arc

lamps or mercury discharge lamps with a laser,

excitation ﬁlter, supercontinuum sources, and

high-power LEDs. Among them, lasers are the

most widely used for more complex ﬂuorescence

microscopy techniques, such as total internal

reﬂection ﬂuorescence microscopy and confocal

microscopy. In contrast, xenon lamps, mercury

lamps, and dichroic excitation ﬁlter LEDs are

ideally used for wide-angle epiﬂuorescence

microscopes. By placing two microlens arrays in

the illumination path of a wide-ﬁeld

epiﬂuorescence microscope (Coumans et al.

2012), highly uniform illumination can be

achieved.

1 Advances in Microscopy and Its Applications with Special Reference to... 11

Fig. 1.3 Diagrams showing the basic principle of ﬂuorescence. (a) Showing excitation of the photon by light energy, (b)

Photon in the excited state, (c) Photon returning to ground state

1.5.2.2 Sample Preparation

in Fluorescence Microscopy

The ﬁrst and foremost criterion for a sample to be

suitable for ﬂuorescence microscopy is that it

must be ﬂuorescent. There are various methods

for creating a ﬂuorescent sample; the main

techniques are labeling with ﬂuorescent dyes or,

in the case of biological samples, ﬂuorescent pro-

tein expression. Alternatively, intrinsic ﬂuores-

cence or autoﬂuorescence of the sample may

also be used. In the life sciences, a ﬂuorescence

microscopy is a powerful tool that enables sensi-

tive and speciﬁc staining of a sample to detect the

distribution of proteins or other molecules of

interest. Thus, a variety of techniques are avail-

able for ﬂuorescent staining of biological samples

of interest (Sanderson et al. 2014).

1.5.3 Fluorophores

Fluorophores/ﬂuorochromes are ﬂuorescent

chemicals that can re-emit the light on excitation.

They are used as a dye to stain structures, probes,

or substrates for speciﬁc enzymes. They cova-

lently attach to the macromolecule under obser-

vation, thus allowing them to be visible under a

ﬂuorescent microscope or spectroscope. Fluores-

cein is the most widely used ﬂuorophore derived

from ﬂuorescein isothiocyanate (FITC). Ideal

ﬂuorophores should be more photostable, less

pH-sensitive, and brighter, and such qualities are

seen in newer-generation ﬂuorophores

(Gustafsson et al. 2008). Since ﬂuorophores

absorb and emit light, the wavelength of the

absorbed light, the efﬁciency of energy transmis-

sion, and the time taken for transmission depend

on the nature of the ﬂuorophore and its interaction

with the environment. Fluorophores are broadly

classiﬁed into four groups based on their molecu-

lar structure and synthetic methods. They are

small organic compounds, proteins and peptides,

synthetic oligomers and polymers, and multi-

component systems (Tsien and Waggoner 1995;

Lakowicz2006).

12 N. B. Pushpa et al.

1.5.4 Immunofluorescence

Immunoﬂuorescence is a technique in micros-

copy widely used in microbiology. It uses

antibodies labeled with ﬂuorescent dyes to illumi-

nate and study the structure under observation

(Lichtman and Conchello 2005). The antibodies

help locate the particular particle throughout the

sample or identify a speciﬁc cell. The region of

contact of the antibody on the antigen is called the

epitope. The antibodies are either covalently

bound to the ﬂuorescent dye or by means of one

more antibody, referred to as primary antibody

and secondary antibody, respectively (Im et al.

2019). Subsequently, the ﬁrst method is called

direct immunoﬂuorescence, and the second is

called indirect immunoﬂuorescence. Immunoﬂu-

orescence provides an essential advantage of

revealing molecules in their indigenous state,

decreasing potential distortion of protein struc-

ture, localization of the protein, and understand-

ing its function using ﬂuorophore tagging

(Mutasim and Adams 2001).

Immunoﬂuorescence is not devoid of

disadvantages, the major drawback being

photobleaching which results in the loss of activ-

ity of the ﬂuorophore (Wäldchen et al. 2015).

This can be reduced by limiting the intensity

and period of light exposure and utilizing

ﬂuorophores of ideal quality, which can resist

photobleaching. Other limitations include restric-

tion to a particular cell and lesser penetration of

ﬂuorophore into the cells (Im et al. 2019).

1.5.5 Components of Fluorescence

Microscope

Aﬂuorescence microscope essentially includes

the following components (Fig. 1.4)—

The light source—is usually a mercury vapor

lamp/Xenon arc lamp/tungsten halogen lamp.

Laser beams in laser scanning microscopes scan

the specimen and create an image. Recent light

source improvement uses bright, single-

wavelength light-emitting diodes (LEDs). Appre-

ciable advantages of LEDs are fast switching

without shutters, increased longevity, and tight

control of wavelength.

Excitation ﬁlter—it ensures the transmission

of only light of the required wavelength. Hence,

it allows the light reﬂected by the dichroic mirror

to illuminate the specimen. Excitation ﬁlter has

limited use when monochromatic or laser light is

for excitation purposes.

Dichroic beam splitter—since the excitation

source is brighter than the emitted light, it is

necessary to have ﬁlters that can block the bright

excitation light. This can be achieved by using

ﬁlters that separate excitation and cast light. By

this arrangement, the specimen’s excitation light

of a shorter wavelength is reﬂected, and light of a

longer wavelength is transmitted to the detector

by the dichroic mirror. Sometimes they can also

be used to send short-wavelength light and reﬂect

the light of longer wavelength (Sanderson et al.

2014).

Emission ﬁlter—when the light passes through

the ﬁlters, there is always a threat of decreasing

intensity. Also, the image brightness is deter-

mined by the light transmitted by the ﬁlters.

Hence it is always essential to have emission

ﬁlters that can only block spurious excitation

light, thus ensuring a bright image of the

specimen.

Filter cube—this is used to align the excitation

and emission ﬁlters in the light path. It directs

light from the excitation source to the specimen

and from the specimen to the detector. Hence, the

orientation of each element is essential within the

ﬁlter cube. It is about a one-inch-sized box

containing a set of excitation ﬁlters, a dichroic

ﬁlter at 45°, and an emission ﬁlter. The set of

ﬁlters included depends upon the ﬂuorescent dye

used. A recent microscope consists of an

automated ﬁlter changer enabling rapid switching

in and switching out ﬁlters from the light path

(Murphy and Davidson 2012)(Fig. 1.4).

1 Advances in Microscopy and Its Applications with Special Reference to... 13

Fig. 1.4 Parts and function

of ﬂuorescence microscope

1.6 Types of Fluorescence

Microscope

With its widespread application, the ﬂuorescence

microscope has become an essential tool for cell

biologists. In this microscope, light from the

source illuminates the specimen to excite the

ﬂuorophore attached. Different types of ﬂuores-

cence have been listed based on the microscope’s

ability to cast a particular region or whole of the

specimen.

1.6.1 Wide-Field Fluorescence

Microscopy

Wide-ﬁeld ﬂuorescence microscopy is Wide-ﬁeld

microscope is ideally suited for viewing 2D

images of the specimen. The most signiﬁcant

advantage of wide-ﬁeld microscopes is the ability

to capture and view an entire specimen at a

glance. Conversely, the image obtained in this

microscope is of less contrast and spatial orienta-

tion owing to diffraction-limited optics and pro-

jection of out-of-focus light on the camera’s

image plane (Pawley 2006). These limitations

can be overcome to a certain extent by the optimal

selection of a specimen that falls entirely under

the focus and with thin sections and by reducing

the numerical aperture of the objective to avoid

out-of-focus light. Other advantages of a vast

ﬁeld microscope are cost-effectivity, simplicity,

reduced photobleaching/phototoxicity, and ﬂexi-

bility (Sanderson et al. 2014).

1.6.2 Confocal Microscopy

Laser scanning microscopes enable thick

specimens and reduce out-of-focus light. In con-

trast to a wide ﬁeld ﬂuorescence microscope, the

light source is a laser- a bright point source. Other

differences are that point source illumination is

sequentially scanned, and the emitted light is

detected using a photomultiplier tube. Also, the

image is compiled pixel by pixel at each point.

The optimal magniﬁcation of the image depends

on the dwell time of the laser spot that remains

over the specimen’s particular site. The greater

the dwell time, the image is formed due to the

increased number of photons being captured.

However, dwell time can simultaneously boost

the chances of photobleaching. Reduction of

focus light is achieved in confocal microscopes

by using a pinhole aperture, as it can exclude light

that is out of focus. However, this is again at the

cost of the reduced intensity of the image

obtained, which is in turn due to reduced photons

captured at that particular dwell time. The other

major drawback of a confocal microscope is

reduced tissue penetration, which can be over-

come using pulsed, long-wavelength laser excita-

tion sources, and two-photon excitation modes.

Two-photon microscopes also concentrate on

photobleaching as there is limited use of

indicators to produce the image. Also, using

more extended wavelength lasers allows for

deeper penetration of tissues (Denk et al. 1994;

Hama et al. 2011).

14 N. B. Pushpa et al.

1.6.3 Total Internal Reflection

Fluorescence Microscopy

The limitations of confocal and two photo

microscopes are reduced axial resolution and

diffraction-limited approach. This resulted in the

invention of a total internal reﬂection ﬂuores-

cence (TIRF) microscope (Axelrod 2001). The

use of total internal reﬂection to illuminate tissues

was ﬁrst advocated by Axelrod (Ambrose 1956).

Here, a glass substrate is used to direct the emitted

light toward the aqueous specimen placed at a

shallow angle. This results in total internal reﬂec-

tion because of the difference in the refractive

index at the glass water interface. The penetration

depth depends on the wavelength of emitted light,

the difference in the refractive index, and the

angle of incident illumination. The major limita-

tion is that this ﬁeld can only excite the

ﬂuorophores, which are just adjacent to the inter-

face, thus resulting in an optical sectioning effect.

This can be addressed by using prism-based TIRF

microscopes, as excitation light bypasses the

objective lens (Brakenhoff et al. 1986;

Mandracchia et al. 2020).

1.7 Applications/Uses

of Fluorescence Microscope

The ﬂuorescence microscope is widely employed

to study structures in biological samples. It

enables the observation of the ultrastructure of

the cell. This technique can also be studied for

the morphology and even the activities within the

cells. With its potential to segregate individual

cell proteins with utmost precision in the midst

of non-ﬂuorescence materials, it is the most pre-

ferred microscopy technique to understand the

dynamic behavior of living cells. Hence it can

detect as low as 50 molecules/mm

. Many

structures can be traced at once with ﬂuorescent

dyes of different colors. This property has led to

the widespread usage of ﬂuorescence microscopy

in neurobiology, physiology, embryology, oncol-

ogy, etc. (Masters et al. 1997).

1.8 Recent Trends and Application

With the advancement in technology, many more

features have been invented in microscopy, thus

extending its applications to different ﬁelds. A

basic ﬂuorescence microscope is a routine instru-

ment for a cell biologist. A few of the recent

inventions include scanning probe microscopy,

ultrasonic force microscopy, digital holographic

microscopy, photoacoustic microscopy, laser

microscopy, etc. The use of a microscope is no

longer limited to observing objects invisible to

the naked eye. It is widely used in the medical

ﬁeld for In-vitro diagnostics, examining forensic

pieces of evidence, 3D imaging, next-generation

sequencing of genetic material, digital assisted

cell morphology, automated digital pathology,

cell sorting, and counting, studying the atomic

structure, etc. When closely placed ﬂuorescent

objects are viewed under a microscope, there is

a greater chance of blur image formation due to

bright blur as each object emits an overlapping

cone of light. Super-resolution microscopy has

helped to overcome the traditional diffraction

limits encountered in optical microscopy (Dani

and Huang 2010). Hence the observation of

objects which are sparsely spaced can yield a

better image, which is the principle behind

photoactivated localization microscopy (PALM)

(Betzig et al. 2006) and stochastic optical recon-

struction microscopy (STORM) (Rust et al.

2006). PALM ﬂuorescence from the individual

ﬂuorophore is separated by temporary separation

of their emission periods. STORM is a super-

resolution variant of the immunoﬂuorescence

technique that uses photo sandwich-able ﬂuores-

cent dyes. Another dynamic approach is the

stimulated emission depletion (STED) micro-

scope. Here indicators are separated by reducing

the excitation spot size. Although the diffraction

limit of microscope optics still exists in this type,

the major advantage of the STED microscope is

using a simple continuous wavelength laser

instead of synchronized pulsed lasers (Hell 2009).

1 Advances in Microscopy and Its Applications with Special Reference to... 15

1.9 Conclusion

The science of microscopy is centuries old.

Inventions with the ability to visualize clearly

and accurately are the key factor that has led to

the evolution of different microscopes. Each

advancement has led to steady improvement in

microscopy, which has revolutionized the ﬁeld of

science, especially medicine. It is indeed micros-

copy that has resulted in the development of

different branches of medicine like histology,

pathology, microbiology, etc. Histopathology

has enabled the accurate diagnosis of various

diseases and timely treatment of the same. Recent

microscopes have made possible three-

dimensional views of various structures/samples

under study. This is aided by updated software

and graphics which can record, store and analyze

complex data. The future of microscopy depends

on the development of technologies that enables

the observer to visualize the sample in three

dimensions with utmost precision. With the appli-

cation of artiﬁcial intelligence, there is more

scope for digitalization and integration by which

the observer can witness the whole process hap-

pening in the entire biological sample. Also,

novel soft wares have been developed to refrain

unwanted signals arising from of focus region of

the sample and disclose the in-focus region of

interest. This will result in enhanced precision

and reliability, thus enabling the observations to

be more reproducible and signiﬁcant. Such

advancements will not only spearhead a new era

of scientiﬁc imaging and fundamentally switch

the way that researchers think when observing

an organism, tissues, and three-dimensional cell

cultures like organoids; they will also optimize

and further enhance what is already existing.

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Visualisation of Host–Pathogen

Communication 2

Amy Dumigan, Ricardo Calderon Gonzalez, Brenda Morris,

and Joana Sá-Pessoa

Abstract

The core of biomedical science is the use of

laboratory techniques to support the diagnosis

and treatment of disease in clinical settings.

Despite tremendous advancement in our

understanding of medicine in recent years,

we are still far from having a complete under-

standing of human physiology in homeostasis,

let alone the pathology of disease states.

Indeed medical advances over the last two

hundred years would not have been possible

without the invention of and continuous

development of visualisation techniques

available to research scientists and clinicians.

As we have all learned from the recent COVID

pandemic, despite advances in modern medi-

cine we still have much to learn regarding

infection biology. Indeed antimicrobial resis-

tant (AMR) bacteria are a global threat to

human health, meaning research into bacterial

pathogenesis is vital. In this chapter, we will

brieﬂy describe the nature of microbes and

host immune responses before delving into

some of the visualisation techniques utilised

in the ﬁeld of biomedical research with a

focus on host–pathogen interactions.W

will give a brief overview of commonly used

techniques from gold standard staining

methods, in situ hybridisation, microscopy,

western blotting, microbial characterisation,

to cutting-edge image ﬂow cytometry and

mass spectrometry. Speciﬁcally, we will

focus on techniques utilised to visualise

interactions between the host, our own bodies,

and invading organisms including bacteria.

We will touch on in vitro and ex vivo

modelling methodology with examples

utilised to delineate pathogenicity in disease.

A better understanding of bacterial biology,

immunology and how these ﬁelds interact

(host–pathogen communications) in biomedi-

cal research is integral to developing novel

therapeutic approaches which circumvent the

need for antibiotics, an important issue as we

enter a post-antibiotic era.

A. Dumigan (✉) · R. C. Gonzalez · B. Morris ·

J. Sá-Pessoa

Wellcome-Wolfson Institute for Experimental Medicine,

Queen’s University Belfast, Belfast, UK

e-mail: a.dumigan@qub.ac.uk;r.calderongonzalez@qub.

ac.uk;b.morris04@qub.ac.uk;j.sapessoa@qub.ac.uk

#The Author(s), under exclusive license to Springer Nature Switzerland AG 2023

E. Abdel Meguid et al. (eds.), Biomedical Visualisation, Advances in Experimental Medicine and

Biology 1406, https://doi.org/10.1007/978-3-031-26462-7_2

Keywords

Cytometry · Host–pathogen interactions ·

Bacteria · Immune system

2.1 Introduction

Microorganisms can be found almost anywhere

on earth playing an important role in ecosystems,

manufacturing industry and human health. They

can also be regarded as human pathogens and

their fast identiﬁcation is crucial in the clinical

setting. To aid in bacterial identiﬁcation, tradi-

tional techniques based on phenotype have been

used routinely for many years. They are based on

morphology, cell wall, growth in different media

or biochemical properties. More recently the

advent of new techniques and their automatiza-

tion allows the use of sequencing or mass spec-

trometry for a more reliable species identiﬁcation.

In this chapter, we describe some of the most

common tests for bacterial identiﬁcation from

staining to molecular methods.

20 A. Dumigan et al.

The major groups of microorganisms studied

in microbiology are bacteria, algae, viruses, fungi

and protozoa. Each of which are found ubiqui-

tously in nature. Most microbes consist of a single

cell and are therefore microscopically ranging

from 20 nm viruses to large protozoans around

5 mm or more in diameter. Viruses are tiny acel-

lular entities, on the border between living and

non-living, only behaving like living organisms

once they gain entry to a living host cell. The

most well-studied of these organisms are bacteria.

Therefore, we rely on visualisation techniques

including but not limited to microscopy to study

the interaction of these organisms with our host

cells in order to understand pathogenicity of dis-

ease with the aim of developing medical

interventions for infection.

Most bacteria can be described as having

either rod, spherical, or spiral morphology with

some also possessing ﬁlaments. Bacteria can be

motile or non-motile and, unlike eukaryotic cells,

bacteria do not have nuclei. Many bacteria are

able to absorb nutrients from their environment,

and some are also capable of making their own

nutrients via processes including photosynthesis

and other synthetic processes we will discuss later

in this chapter. Herein we will focus on the study

of bacterial infection biology, meaning the study

of bacterial interaction with mammalian host

cells. Although bacteria can interact directly and

indirectly with all mammalian cells, we will focus

on interaction with the main drivers of host

defence, i.e. the immune system.

2.1.1 The Immune System

The immune system is a multi-faceted and com-

plex collaboration of cells acting to protect the

host from infection and maintain the health of

tissues. The main functions of the immune system

are to act as a barrier, recognise pathogens and

initiate immune responses to restore homeostasis

and the health of the host. Epithelial surfaces

provide the initial defences against invading

pathogens via mechanical, chemical and

microbiological methods. Tight junctions

between epithelial cells of the skin and linings

of the lungs, gut and urogenital tracts act as a

mechanical barrier to the external environment.

The presence of mucus and internal commensal

ﬂora also prevents the passage and establishment

of pathogens across epithelial barriers. The

importance of the epithelia becomes apparent

when the barrier is breached by wounds, burns

or loss of epithelial integrity and leads to an

increased risk of infection (Murphy et al. 2008).

The immune system consists of innate and

adaptive immune responses which can be

inﬂuenced by factors such as genetics and the

environment (Janeway et al. 2001). All multi-

cellular organisms possess innate-like immunity

including fungi, plants, insects and higher

organisms (Beutler 2004). However, only

vertebrates beneﬁt from an adaptive immune sys-

tem and immunological “memory”(Cooper and

Alder 2006). The innate immune response is

immediate, consistent and broad acting and

activates adaptive immune responses. Con-

versely, the adaptive immune response is delayed

and slow acting upon primary encounter with a

pathogen, yet is highly speciﬁc and provides

immunological memory, allowing a rapid

response upon secondary exposure to the same

pathogen (Lee and Mazmanian 2010). Cells from

both innate and adaptive systems coordinate

responses via cell–cell contact or production of

cytokines or chemokines (Getz 2005), mounting

effective and tightly controlled responses while

preventing further damage to tissues. Primary

immunodeﬁciency disorders (PID) are a group

of heterogeneous illnesses caused by defects in

immune system function. Patients with PID are

much more susceptible to autoimmune disorders,

malignancy and recurrent infections (Raje and

Dinakar 2015). Indeed, bacteria have evolved

strategies to circumvent healthy immune

responses for their own beneﬁt.

2 Visualisation of Host–Pathogen Communication 21

2.1.2 Innate Immunity

The innate immune system has evolved over

millennia to recognise and destroy invading

pathogens (Carrington and Alter 2012). Innate

immunity provides an immediate response

towards foreign antigens. Several factors can

affect the efﬁcacy of innate immune response

including age, health, genetics and lifestyle. The

main function of innate immunity is to detect and

eradicate cells expressing non-self-antigen and is

the ﬁrst active line of defence against infection.

The innate system is largely dependent on cells of

the myeloid lineage. Myeloid-derived cells

develop in the bone marrow or specialised

tissue-resident cells and include macrophages,

dendritic cells (DCs), neutrophils, basophils,

eosinophils and natural killer (NK) cells of the

lymphoid lineage. These are all specialised cells

with individual functions that work together

towards the destruction and/or clearance of for-

eign pathogens and damaged cells (Murphy et al.

2008).

Macrophages are distributed throughout the

body and are specialised within tissues,

i.e. ﬂattened Küpffer cells of the liver and

microglial cells of the central nervous system

(CNS) (Beutler 2004). In addition to the clearance

of dead cells and debris in tissue homeostasis,

macrophages and DCs (together with neutrophils)

have numerous roles in innate immunity, activa-

tion of adaptive immunity, inﬂammation,

response to damage and tissue repair.

The main role of macrophages is the phagocy-

tosis and destruction of non-self-antigen, dead

cells and debris. To elicit effective clearance of

pathogens, Macrophages must ﬁrst recognise

them via surface receptors. The type of phagocy-

tosis is dependent upon the target and its location

although generally the binding of target antigen to

its receptor leads to adherence of the antigen to

the macrophage plasma membrane; this

stimulates contraction of actin and myosin

ﬁlaments leading to extension of pseudopods

around the non-self-matter. As further receptors

engage, the foreign antigen is encapsulated in a

vacuole known as a phagosome. Cytoplasmic

lysosomes fuse with the phagosome and release

their contents, which include reactive oxygen

species and nitric oxides, leading to the destruc-

tion of the foreign antigen. Degradation of the

target reveals new ligands which are processed

and presented on the cell surface via major

histocompatability complex (MHC) (Underhill

and Goodridge 2012).

Without even realising it, our bodies are in a

constant ﬂux, our cells are communicating with

one another to elicit strategic and tightly con-

trolled responses in order to protect us from a

plethora of microscopic particles each day. Our

immune system is made up of specialised cells, all

interacting with one another directly or indirectly

to orchestrate a response to invading pathogens

during infection. In the ﬁeld of host–pathogen

interactions, we combine techniques and knowl-

edge from both microbiology and immunology to

uncover mechanisms utilised by pathogens to

circumvent destruction by immune cells, and ulti-

mately how to prevent that therapeutically. In

order to do this we must utilise several techniques

to assess all aspects of this interaction.

Interestingly some commensal or “good bacte-

ria”found in our bodies, or in the environment,

can become problematic or pathogenic whenever

they enter areas of our bodies which are not

adapted to their presence. For example, when

certain bacteria which are harmless when in the

gut, get into the lungs, can lead to pneumonia or

into the blood leading to septicaemia. One such

example of an opportunistic pathogen is Klebsi-

ella pneumoniae.

22 A. Dumigan et al.

2.1.3 Klebsiella Pneumoniae

and Antimicrobial Resistance:

The Problem

K. pneumoniae is represented by the “K”in

ESKAPE pathogens, the six most signiﬁcant

causes of drug-resistant hospital infections and

is recognised by the Centre for Disease Control

and Prevention, World Health Organisation, and

the European Union as a signiﬁcant threat to

global health due to the increasing rates of anti-

microbial resistance (Rice 2008;WHO2014).

A report published in 2022 has shown that,

globally, greater than 600,000 deaths were

associated with, and approximately 200,000

deaths were attributable to K. pneumoniae in

2019 (Murray et al. 2022). K. pneumoniae is

intrinsically resistant to ampicillin, but extended

spectrum beta-lactamase genes, resistance genes

against carbapenemases and most recently colis-

tin have now also been reported, leaving ever-

reducing therapeutic options (Petrosillo et al.

2019; Aris et al. 2020).

Globally, the third highest mortality rates due

to multi-drug resistant infections are either

associated with or attributable to K. pneumoniae

(Murray et al. 2022). Between 2014 and 2017 the

total number of bacteraemia cases caused by

Klebsiella species in England increased by 12%

and Northern Ireland had the highest rates (Public

Health England 2020). Trends for 2011–2015

show increasing resistance to third-generation

cephalosporins, ﬂuoroquinolones,

aminoglycosides, and extended spectrum beta-

lactams in England, while at the minute resistance

to carbapenems remains relatively uncommon

(Public Health England 2020). K. pneumoniae

has been described as a “canary in the coalmine”

as several antibiotic resistance genes have been

ﬁrst identiﬁed in Klebsiella species (Holt et al.

2015; Wyres et al. 2020). As a promiscuous

donor and recipient of plasmids containing anti-

microbial resistance (AMR) genes,

K. pneumoniae, and other Gram-negative bacte-

rial species are accumulating AMR genes, further

reducing the resources there are to treat these

infections (Holt et al. 2015; Wyres et al. 2020).

With the seemingly exponential rise in AMR

bacterial species worldwide, it has never been

more important to understand the host–pathogen

interactions of bacteria versus the host. Indeed the

need for the development of effective

immunotherapeutics which reduce or eradicate

dependency on antibiotics is incredibly impor-

tant. Without this, we are facing a post-antibiotic

era. Using both gold standard and cutting-edge

techniques we can uncover the pathogenesis of

disease and develop novel therapeutics.

Visualisation of interactions is an integral aspect

of research and spans from simple staining to

mass spectrometry, ﬂow cytometry, and CYTOF

technologies.

2.1.4 In Situ Visualisation of Host–

Pathogen Communications

Using ﬂuorescent microscopy, immunohisto-

chemistry, mass spectrometry and ﬂuorescent in

situ hybridisation techniques we can ascertain an

abundance of information regarding bacterial

infection and host responses. Herein we will

describe the applications of each method.

2.1.4.1 Microscopy

Lenses have been used to start ﬁres and correct

vision since around the year 1300. However, it

was not until 300 years later that the Dutch and

Italian spectacle makers combined lenses to visu-

alise far-away objects, a discovery that led to the

ﬁrst microscope. The visualisation of cells as we

now know then was not until Antoni van

Leeuwenhoek, a Dutch fabric merchant utilised

rudimentary microscopes to identify motile or

“animate”particles (now knowns as cells) from

“inanimate”ones, namely sperm, bacteria, and

protozoa (Shapiro 2003). The term “cells”was

not used until the mid-1800s, when the develop-

ment of condensers and the utilisation of multiple

lenses improved resolution and allowed

identiﬁcation of ﬁne cellular structures could be

differentiated from artefacts (Shapiro 2018). By

the late 1800s Louis Pasteur, Robert Koch and

others developed the cell theory, combining their

work visualising the metabolic diversity and path-

ological potential of microorganisms. Indeed,

Rudolf Virchow embodied the efforts of many

pathologists to understand disease at a molecular

level with his quote “Omnis cellula e cellula”

(Schultz 2008).

2 Visualisation of Host–Pathogen Communication 23

2.1.4.2 Stains and Dyes

Visualisation of the intricate cellular structures,

morphology and cell-to-cell interactions of

tissues and cells was impossible until the devel-

opment of staining techniques. Indeed, it was not

until around the 1860s that it was determined that

different coloured organic dyes had chemical

afﬁnities that could bind to a variety of cellular

components with varying degrees of intensity.

This work was led by Paul Ehrlich during his

medical education (Kahr et al. 1998). He discov-

ered that the use of synthetic dyes could stain

specimens and allow visualisation of different

cellular components and thus allowed identiﬁca-

tion of different cell subsets within a mixed sam-

ple. In 1882, Ehrlich joined forces with Robert

Koch to develop a stain to detect Mycobacterium

tuberculosis (Mtb), a process which was further

developed by others to result in the e Ziehl–

Neelsen (ZN) stain, which remains a gold stan-

dard technique to detect Mtb (Shapiro 2018).

Most cells and microorganisms lack colour

and contrast and are impossible to observe or

properly characterise morphology under the

microscope without the use of staining. In clinical

settings, a specimen can be prepared as a wet

mount (drop of liquid on a slide or smear of a

tissue in a drop of liquid such as water) or ﬁxated

(attached cells to a slide). Fixation can involve

heat or use of chemical ﬁxatives (methanol, form-

aldehyde, ethanol and glutaraldehyde being the

most common), killing the microorganisms in the

specimen while preserving their integrity. After

ﬁxing, cells are usually stained to apply colour

and facilitate visualisation (Howat and Wilson

2014).

Staining can be done using simple stains that

colour all the specimens regardless of only one

type of organism being present. If the coloured

ion (chromophore) is positively charged it is a

basic dye and binds negatively charged

components such as cell walls (positive stain).

Examples include crystal violet, malachite

green, methylene blue and safranin. If the chro-

mophore is negative, the stain is acidic and tends

to be repelled by negatively charged cell walls of

microbes (negative stain). Examples include

eosin and rose Bengal (Franco-Duarte et al.

2019).

Differential staining on the other hand

involves the use of multiple stains giving different

colours based on the interaction with the dye and

the microorganism’s physio-chemical properties.

The most common example of this type of stain is

Gram staining. In Gram staining, ﬁrst crystal vio-

let is applied followed by iodine to ﬁx the dye and

ethanol wash (at this point Gram-negative bacte-

ria lose the colour while Gram-positive remain

blue or purple). As a counterstain safranin is

used which stains Gram-negative bacteria red.

Another example is the acid-fast technique that

allows to distinguish acid-fast bacteria such as

Mycobacterium. Cells are stained with

carbolfuchsin (red) using a lipid solvent and

washed with a dilute acid-alcohol solution

which will be washed away from non-acid-fast

bacteria that take up methylene blue afterward.

Other stains allow identiﬁcation of bacterial

structures such as ﬂagella (using metals) or spores

(using malachite green) (Chauhan and Jindal

2020). Examples are depicted in Table 2.1.

2.1.5 Biochemical Tests

Different bacteria have different biochemical

requirements, and these have been used tradition-

ally in the clinic for the classiﬁcation of bacteria

(sometimes up to species level). They rely on

their carbon or metabolic usage capabilities and

can be done in test tubes or using commercially

available kits that allow to screen for a panel of

biochemical reactions and their comparison to a

database (Biolog using multiwell plates with the

main carbon and nitrogen metabolites for growth,

API-20A system using dry powder substrates or

VITEK automated broth analysis for instance).

24 A. Dumigan et al.

Table 2.1 Examples of commonly used stains to determine physio-chemical characteristics of microorganisms

Stain type Dyes Purpose

Simple

stains

Basic Methylene blue, crystal violet, malachite green,

safranin.

Stain negatively charged structures

Acidic Eosin, rose Bengal, congo red. Stain positively charged structures.

Negative India ink, nigrosine. Stains background, not sample.

Differential

stains

Gram Uses crystal violet, Gram’s iodine, ethanol and

safranin.

Distinguishes cells by their cell wall

(Gram-positive are purple, Gram-

negative are pink).

Acid-fast Basic fuchsin, acid-alcohol and methylene blue. Distinguishes acid-fast bacteria such

as Mycobacterium (red) from

non-acid fast cells (blue).

Endospore Heat with malachite green and counterstain with

safranin.

Endospores appear green while other

structures are red.

Capsule Negative staining using India ink that stains the

background and capsule stays clear if a

counterstaining is used to stain the cell

Capsules appear clear or halos while

background is dark.

Common biochemical tests include:

•Catalase test (detects catalase, an enzyme that

catalyses the release of oxygen from hydrogen

peroxide present in most aerobic or facultative

anaerobic bacteria such as Staphylococcus)

•Coagulase test (used to differentiate Staphylo-

coccus aureus from coagulase-negative

Staphylococciby inoculation with plasma)

•Oxidase test (detects the presence of cyto-

chrome oxidase in Pseudomonas and Vibrio,

which is absent in Escherichia coli,Klebsiella

or Salmonella)

•Indole test (detects the ability to degrade tryp-

tophan and is used to distinguish Enterobac-

teriaceae such as E. coli that is positive from

Klebsiella that is negative)

•Citrate test (ability to use citrate as the only

carbon source, distinguishes citrate-positive

Klebsiella from E. coli)

•Urease test (detects the ability to degrade urea

and produce ammonia and can distinguish

urease-positive Proteus from urease-negative

E. coli for instance) (Fig. 2.1)

Other tests based on biochemical properties

could be the identiﬁcation of bacteria based on

their lipid composition using FAME (fatty acid

methyl ester) or PFLA (phospholipid-derived

fatty acids) analysis or the identiﬁcation of bacte-

ria based on their unique mass spectrum using

MALDI-TOF) (Chauhan and Jindal 2020).

2.2 Selective

and Differential Media

Another method commonly used to identify bac-

terial species is the use of selective media. This is

when the growth medium is either supplemented

with growth factors which are beneﬁcial to the

health of the bacteria of interest or includes

growth inhibitors which will remove extraneous

species. This method allows the identiﬁcation of

closely related microorganisms.

Media may be supplemented with antibiotics

which allows only microorganisms resistant to

that antibiotic to grow. Examples of differential

media include those that have indicators or

nutrients that allow a certain biochemical charac-

teristic of a microbe to become apparent (in citrate

agar K. pneumoniae appears as yellow colonies

because they metabolise citrate while E. coli

appears as blue colonies). Table 2.2 depicts

examples of common media.

d e

2 Visualisation of Host–Pathogen Communication 25

Fig. 2.1 Identiﬁcation of Staphylococcus aureus based on phenotypic and biochemi-

cal tests. (a) Colonies showing pigmented smooth colonies, catalase-positive and

Gram-positive cocci. (b) Biochemical test strips. (c) Identiﬁcation of the biochemical

test. ( ) Coagulase test. ( ) Test of resistance to the antibiotic novobiocin. This ﬁle is

licensed under the Creative Commons Attribution-Share Alike 4.0 International

license. https://creativecommons.org/licenses/by-sa/4.0/legalcode

26 A. Dumigan et al.

Table 2.2 Examples of selective and differential growth media commonly used to identify microorganisms

Type Media name Types of organisms distinguished

Selective YM Yeast and mold

MacConkey Agar Gram-negative bacteria

Mannitol Salt Agar Gram-positive bacteria

Differential Blood agar Becomes transparent in hemolytic strains such as Streptococcus pyogenes

MacConkey Lactose fermentation

X-gal plates Lac operon mutants

2.3 Molecular Methods

Not all microorganisms are culturable in labora-

tory conditions and standard culturing and bio-

chemical tests are time-consuming. Molecular

techniques are more advantageous since they are

rapid, more sensitive and more speciﬁc, allowing

as well to identify strains that are difﬁcult to

culture. They include 16S sequencing, RT-PCR,

random ampliﬁed polymorphism

deoxyribonucleic acid (RAPD), restriction frag-

ment length polymorphism (RFLP), whole-

genome sequencing (WGS) sequencing and

MALDI-TOF (Franco-Duarte et al. 2019).

16S rRNA PCR is one of the most commonly

used molecular methods. It relies on the fact that

this rRNA is highly conserved amongst bacteria

but has variability regions that can be used to

identify speciﬁc species. It involves the PCR

ampliﬁcation of the 16S rRNA gene, sequencing

and comparison to a database. It can be applied to

a variety of samples but involves pre-processing

as some samples can have compounds that inhibit

the PCR reaction. RAPD-PCR on the other hand

involves the ampliﬁcation of random sequences

in template bacterial DNA using short primer

sequences, resulting in a proﬁle for identiﬁcation

that can be compared to a database. It has the

advantage of allowing the identiﬁcation of bacte-

ria that have not been sequenced and requires no

previous DNA isolation. RFLP uses restriction

enzymes that can cut PCR products into different

fragments generating a unique pattern. WGS has

become more affordable and can be useful both in

bacterial genotyping but also in antimicrobial

resistance allowing to investigate clinical

outbreaks. In MALDI-TOF MS, the proteins of

a bacterial species are analysed by creating

a mass:charge ratio pattern that can be compared

to a library of strains (Adzitey et al. 2013).

2.4 Haematoxylin and Eosin

Ehrlich experimented with acidic (able to stain

cell cytoplasm) and basic (nucleic) dyes. One

such dye was developed from logwood

(Hematoxylon campechianum) extract, discov-

ered by Spanish explorers in 1502 (Kahr et al.

1998), leading to the development of

Haematoxylin and Eosin (H&E) staining tech-

nique. H&E staining, arguably ﬁrst utilised nearly

200 years ago in 1830 (von Waldeyer 1863),

though nearly 200 years old, is still considered

the gold standard staining technique used by

pathologists and researchers globally today.

Indeed, millions of microscopy slides are

prepared daily to be viewed by pathologists for

clinical diagnosis (Titford 2005). When bacteria

are present in large numbers, in an abscess for

example, they appear as blue-grey granular mass

using H&E stain. Often in research, when

identifying differences in pathogenicity of bacte-

rial strains, it is more informative to assess host

responses using H&E staining.

To better understand how K. pneumoniae

interacts with immune response, Dumiganb

et al. developed a novel whole lung ex vivo infec-

tion model using porcine lungs. Histological anal-

ysis of porcine tissues utilised in a whole lung

ex vivo lung perfusion model of infection

(Dumigan et al. 2019) was carried out based on

parameters of acute respiratory distress syndrome

(ARDS) in animal models as deﬁned by the

American Thoracic Society. Pathogenic

hallmarks of lung injury include the thickening

of alveolar septa and inﬁltration of proteinaceous

debris, red blood cells (haemorrhage), and

immune cells, including neutrophils, into the

alveolar space (neutrophilic alveolitis) (Matute-

Bello et al. 2011). Analysis of lung sections

stained with H&E revealed signs of injury in the

lungs infected with the opportunistic pathogen

Klebsiella pneumoniae, although injury was

more severe in those lungs infected with the

non-attenuated clinical strain Kp52145

(Fig. 2.2a). This was further conﬁrmed by analy-

sis of alveolar septal thickness (Fig. 2.2b). Infec-

tion with an attenuated mutant strain lacking

polysaccharide capsule (a major virulence factor

of Klebsiella pneumoniae) induced signiﬁcantly

enhanced alveolar septal thickening compared to

that of PBS controls; however, this damage was

signiﬁcantly reduced compared to that of

Kp52145-infected lungs (Fig. 2.2b).

2 Visualisation of Host–Pathogen Communication 27

Fig. 2.2 Porcine EVLP model recapitulates clinical

hallmarks of K. pneumoniae-induced pneumonia. (a)

Haematoxylin and eosin staining of porcine lung samples

(magniﬁcation, ×400) from lungs mock-infected (PBS) or

infected with Kp52145 and strain 52145-Δwca

.(b)

Alveolar septal thickness was measured using ImageJ

software. Each dot represents an average of three alveolar

thicknesses per image, corresponding to three sections per

lung across three experimental replicates from lungs

mock-infected (PBS) or infected with Kp52145 and strain

52145-Δwca

.(c) Intra-alveolar haemorrhage was scored

per image whereby 0, 1, 2, and 3 represent none, mild,

moderate, and severe levels of red blood corpuscles within

the alveolar space from lungs mock-infected (PBS) or

infected with Kp52145 and strain 52145-Δwca

.(d)

Number of nucleated cells evident in the alveolar space

per image from lungs mock-infected (PBS) or infected

with Kp52145 and strain 52145-Δwca

. Statistical anal-

ysis was carried out using one-way ANOVA with

Bonferroni correction. Error bars indicate SEM. (Dumigan

et al. 2019). Image shared with permission of

corresponding author Prof Jose Bengoechea

Using scoring, we can glean further quantita-

tive information from histology. The presence of

intra-alveolar haemorrhage was assigned a score

of 0, 1, 2, or 3 based on a semiquantitative assess-

ment of none, mild, moderate, or severe. Scoring

conﬁrmed signiﬁcantly enhanced haemorrhage in

lungs infected with Kp52145 compared to that in

the PBS-mock-infected lungs and the lungs

infected with the cps mutant (Fig. 2.2c).

Haemorrhage was accompanied by the presence

of inﬂammatory immune cells within the alveolar

space. The number of nucleated cells in the alve-

olar space was quantiﬁed, and it was signiﬁcantly

higher in the lungs infected with Kp52145 than in

those infected with the cps mutant or PBS-mock

infected (Fig. 2.2d).

28 A. Dumigan et al.

In terms of methodology in this model, tissue

sections (×1cm

) were collected from the cra-

nial, middle, and caudal lobes of each lung ﬁxed

in 10 ml 10% formalin. After a minimum of 48 h

at room temperature, samples were processed for

parafﬁn embedding, sectioning, and

haematoxylin and eosin staining. Samples were

imaged using a DM5500 Leica vertical micro-

scope at a magniﬁcation of ×200. Alveolar septal

oedema was quantiﬁed by measuring the alveolar

septal thickness with ImageJ software, whereby

three measurements of the thickest septa were

acquired per image and averaged, and 30 images

were acquired, whereby 10 images were acquired

per section and 3 sections per lung. Alveolar septa

adjacent to a blood vessel or airway were

excluded due to normal thickening resulting

from collagen deposition. Intra-alveolar

haemorrhage and the presence of intra-alveolar

mononuclear cells and proteinaceous debris

were also recorded. Histological scores were

assigned based on parameters described previ-

ously (Matute-Bello et al. 2011). Haemorrhage

was scored as follows: 0, none; 1, mild; 2, moder-

ate; and 3, severe. Proteinaceous debris scored as

follows: 0, none; 1, protein present; and 2, abun-

dant presence of protein in alveolar spaces. The

number of nucleated cells within the alveolar

space was counted and presented as intra-alveolar

leukocytes. Five images were scored per section,

with three sections per lung at a magniﬁcation of

×400 (Dumigan et al. 2019).

The use of H&E staining in this model allowed

us to determine that each of the major hallmarks

of human disease was reproducible in our porcine

model of disease. In addition, we were able to

determine that the model was sensitive enough to

test alternative bacterial strains and therefore

draw conclusions for further research (Dumigan

et al. 2019).

2.5 Fluorescence Microscopy

Microscopy is an important tool in following

host–pathogen interactions. It allows the

visualisation of the complex interactions between

host and pathogen and ascertains how the subcel-

lular localisation could be important in shaping

those interactions. It has been evolving since the

seventeenth century and now besides allowing us

to observe ﬁxed or frozen tissues it allows even to

observe at the molecular level.

Fluorescence microscopy takes advantage of

the fact that many compounds or proteins have

ﬂuorescence properties allowing for visualisation

by tagging single molecules with these

ﬂuorophores. Another option is immunolabeling

whereby a protein is tagged with an antibody

molecule and a cognate ﬂuorescent antigen. The

advent of confocal microscopy which allows

high-resolution and contrast images has improved

visualisation. Furthermore, super-resolution

approaches such as stimulated emission depletion

microscopy (STED) or super-resolution

structured illumination microscopy (SR-SIM)

have allowed to observe immune responses at

the molecular level (Wen et al. 2020).

2.6 Fluorescence In Situ

Hybridisation (FISH)

Starting with the ﬁrst in situ hybridisation (ISH)

experiments in 1969 and the ﬁrst uses of

non-radioisotopic probes in the mid-1970 s to

the current implementation of microﬂuidics

(Huber et al. 2018), FISH has become a common

technique used both on research and clinical

diagnostics.

The basic principle of this method is the ISH of

a known-sequence probe with a speciﬁc sequence

inside the cells. Both probe and target can be

either RNA or DNA. Multiple uses of FISH tech-

nique have been described (Volpi and Bridger

2008), but the processing steps are the same for

all of them, and independently that the target is a

cytological, histological or a whole-mount prepa-

ration (Young et al. 2020).

The ﬁrst step for a FISH experiment is the

design of the probes to use. Regardless of any

other aspect, they need to be complementary to

the target sequence we want to detect. Their size

can vary, as well as the ﬂuorescent molecule that

they have attached and that will allow their detec-

tion. Larger probes, between 500 and 1500 bases,

are useful when high sensitivity is required (also

being less expensive), while shorter ones offer

high speciﬁcity. That is the case of the Stellaris™

probes, with just a few dozens of nucleotides

length, which allow the detection of individual

molecules of mRNA (Orjalo et al. 2011).

2 Visualisation of Host–Pathogen Communication 29

Once we have the appropriate probes, we can

perform the ISH (usually after ﬁxation and

permeabilisation steps, to preserve the cellular/

histological structure and facilitate the entry of

the probes into the cells). For this, optimal

conditions have to be assured, paying attention

to several factors such as temperature, time, pH,

salt concentration, etc. One critical factor is the

addition of formamide. The melting temperature

) of DNA is deﬁned as the temperature at

which half of the DNA strands are present as

single strands. DNA denaturalisation is required

prior to the hybridisation with the probes, and as

this is usually obtained at high temperatures, there

is the risk of damaging the cell/tissue structure.

The use of formamide reduces the T

(McConaughy et al. 1969), linearly by 2.4–2.9 °

C per mole of formamide (Blake and Delcourt

1996). Once the hybridisation has been done,

several washes in order to reduce the background

signal both from the unbound probes and

autoﬂuorescence or light scatter caused by the

tissue (Richardson and Lichtman 2015). With

the samples prepared and mounted, all that

remains is visualising the samples in a ﬂuores-

cence microscopy and, if required, performing

quantitative analysis.

2.7 Flow Cytometry

It was in the 1880s when the term “cytometer”

appeared, used to describe a device which could

count the number of cells in a particular volume.

At this time leukaemia and anaemia could be

identiﬁed, and although the cause was unclear, it

was understood that changes in cell morphology

and number over time could indicate the clinical

outcome of the patient. Though a cytometer

describes the device, “cytometry”is the process.

As the cells analysed at this time often came from

human blood “haem”, an easily obtained sample,

led to the birth of “haemocytometer”. Microscopy

remained the main instrument to assess cell num-

ber and morphology until the 1950s and “ﬂow

cytometry”would not come around until

the 1970s (Harris 1999; Shapiro 2018). In 1953

the ﬁrst ﬂow cytometer was disclosed, using the

Coulter Principle to assess cell numbers in a sus-

pension. The ﬁrst cell sorter using a similar prin-

ciple with addition of droplet-based methods for

subsequent cell puriﬁcation arrived in 1965

(Fulwyler 1965). Flow cytometry, as we know it

today, could not have come about without the use

and development of synthetic dyes to stain

samples. This was an essential step as light scat-

tering and absorption by cells is insufﬁcient to

allow visual discrimination of internal structures.

In 1968 absorption-based methods for ﬂow

cytometry using ﬂuorescent antibodies emerged,

a pivotal development meaning speciﬁc cell types

could be identiﬁed in a given population. Cell

sorting and ﬂow cytometry rapidly became popu-

lar, especially with the development of monoclo-

nal antibodies (Köhler and Milstein 1975).

Over the last 70 years ﬂow cytometry and cell

sorting platforms have beneﬁted immensely from

developments in hardware and reagents.

Developments in device design have reduced

device size, costs, and improved ﬂexibility and

ergonomics. Flow cytometry allows multi-

parametric cell analysis; it can be used to deter-

mine the quantity of a given cell type, cell sorting

and analysis of biomarker expression. Presently, a

standard ﬂow cytometer combines ﬂuidics, optics

and software to detect particles using ﬂuorescence

and morphological characteristics –namely size

and granularity. It is a statistically powerful tech-

nique used to characterise heterogeneous

populations from a single-cell suspension. This

single-cell suspension can be whole blood, or

adherent cell lines or solid tissue which has been

dissociated to a single-cell suspension. For solid

tissues, this can be done by homogenising using a

handheld homogeniser (much like a small

blender), or with mechanical force aided by

digestive enzymes. Tissue suspensions often

require ﬁltering using cell strainers or

nylon mesh.

30 A. Dumigan et al.

The ﬂuidics system allows the single-cell sus-

pension to be aligned in an orderly stream via

hydrodynamic or acoustic focusing. This means

that when a single-cell suspension is passed

through the ﬂow cell, the cells are focused into a

central core stream that is surrounded by an outer

sheath of higher pressured ﬂuid. As the cell sus-

pension is passed through the ﬂow cell it

intercepts laser beams, they scatter the light and

emit ﬂuorescence, which is picked up by

detectors and presented on screen as a dot. The

position of the dot on the ﬂow plot will indicate

the marker expression on a given cell.

We know from previous research mentioned

earlier, that immune cells are specialised, mean-

ing they each express markers of their lineage.

Antibodies are raised against these antigens and

labelled with a ﬂuorescent tag. Depending on the

number of lasers a cytometer possesses, we can

look at several parameters at once to delineate a

particular cell type and assess a given line of

investigation. Cell suspensions are generally

prepared by ﬁrst staining markers on their cell

surface (for instance Ly6C is a marker for cells

of the myeloid lineage, including macrophages

and DCs, but not neutrophils). After cell surface

markers have been stained with antibodies tagged

with ﬂuorophores, cells are then ﬁxed and

permeabilised in order to stain intracellular

components such interferon-gamma (IFNg) a

proinﬂammatory cytokine. Before washing and

analysis using a ﬂow cytometer (Fig. 2.3).

We used this technique to assess the number

and activation status of NK cells between wild

type (WT) and genetically modiﬁed mice lacking

receptors for Type I IFNs (IFNAR

-/-

), as shown

in Fig. 2.3. Type I IFN signalling induces NK cell

accumulation and activation in lungs during

K. pneumoniae infection (Ivin et al. 2017).

Flow cytometry is an incredibly useful tool

employed by research and clinical labs globally

and is only set to become a more powerful tool in

the future.

2.8 Imaging Flow Cytometry (IFC)

Imaging ﬂow cytometry (IFC) is a relatively new

technology combining the conventional beneﬁts

of non-imaging ﬂow cytometry to identify,

i.e. analyse and quantify single cells in a heterog-

enous population, with additional imaging IFC,

we can determine the size, shape, and structure of

a cell. This has inherent beneﬁts as morphological

evidence can help determine the stage and pro-

gression of disease, improving biomedical

research and clinical decisions to design

personalised patient treatment strategies (Lei

et al. 2018). IFC has rapidly become an

established cytometric analysis tool in a wide

range of biological research including, but not

limited to, microbiology, immunology, and stem

cell biology (Lei et al. 2018; Goda et al. 2019).

This technique provides spatially registered, mor-

phological, and quantitative data for each, and

every event captured, meaning each cell can be

characterised in detail providing an image of each

cell as well as cytometric evaluation within pure

or heterogenous populations. IFC is effective for

the detection of cell death, DNA damage/repair,

quantiﬁcation of speciﬁc proteins, and peptides. It

can also be combined with ﬂuorescent in situ

hybridisation. In addition, computational biology

can also be utilised in combination with IFC

(Eulenberg et al. 2017). This detailed analysis

provided by IFC allows us to better understand

cell-to-cell interactions in a physiologically rele-

vant setting. Indeed, this technology has a pleth-

ora of potential clinical uses including stage and

progression of blood-born cancers and the identi-

ﬁcation of infectious diseases (Goda et al. 2019).

Signiﬁcant expansion of IFC capabilities is

expected in the next decade or so, especially

with the emergence of microﬂuidics and its inte-

gration into IFC systems. Microﬂuidics rather

than traditional capillary-based ﬂow cytometry

provides greater versatility in terms of

multiplexing and automation, thus providing

greater scope for the preparation, analysis and

manipulation of cells (Goda et al. 2019).

2 Visualisation of Host–Pathogen Communication 31

+24 h

Inoculum

Surface staining Intracellular

Staining

FACS

NK1.1

+NK cells

% of CD45°

cells/lung

12 h 12 h

2×105

4×105

6×105

8×105

PBS NK 1.1

3,56

3,25

4,4

2,52

[log10fl.]

NK1.1

+NK cells

CD3

Ifnar1-/-

Fig. 2.3 Schematic representation of ﬂow cytometry

analysis of murine tissues. Mice were infected with

K. pneumoniae or PBS controls, at end of infection,

lungs are harvested and homogenised before red cells are

removed and cell suspension stained with surface

expressed markers (blue squares), cells were then ﬁxed

and permeabilised before intracellular targets are stained

(orange square) followed by analysis by ﬂow cytometry.

Shown here are ﬂow plots depicting number of NK cells

within WT and IFNAR-/-animals from Ivin et al.

(2017). Image included with permission from

corresponding author Prof Jose Bengoechea

2.9 ELISAs

Infections can lead to an imbalance of the host

immune response and therefore it is important to

use immunoassays to determine to what extent

that imbalance is affecting the clinical outcomes.

Enzyme-linked immunosorbent assay

(ELISA) is a sensitive immunoassay used to

detect antibodies, antigens, proteins,

glycoproteins and hormones. It is a microwell

plate-based method that relies on the binding of

an antigen to a target antibody generating a

detectable signal, either chemiluminescent, ﬂuo-

rescent or colorimetric.

In Klebsiella pneumoniae infection research

our laboratory has used this method to detect a

natural block of the immune system upon infec-

tion. This pathogen dampens the activation of

inﬂammatory responses by inhibiting the activa-

tion of the NF-κB pathway and reducing levels of

IL-8 released by the immune system (Tomás et al.

2015).

2.10 Western Blotting

Western blotting or immunoblotting was

introduced by Towbin in 1979 and is used to

separate and identify proteins (Kurien and

Scoﬁeld 2015). The technique involves gel elec-

trophoresis that separates a mixture of proteins

based on molecular weight which is then trans-

ferred to a membrane incubated with antibodies

speciﬁc to the protein of interest. To prepare

samples for running on a gel, cells and tissues

are lysed to release the proteins of interest and

solubilised so they can be migrated individually

on the gel. Antibodies recognise a small portion

of the protein of interest (the epitope) proteins

need to be denatured (unfolded) to allow binding.

An anionic detergent and heat are applied for

denaturing the proteins. Blocking the membrane

prevents the non-speciﬁc background binding of

the antibodies. To visualise the protein of interest,

a primary protein-speciﬁc antibody is applied

followed by a secondary antibody for detection

and visualisation. Immunoblotting is routinely

used in the lab to detect immune responses to

bacterial infection by probing common proteins

in signalling pathways affected by the pathogen

(Wen et al. 2020).

32 A. Dumigan et al.

2.11 Visualisation of Bacterial

Factors

Bacteria range in size from 0.5 to 5 micrometres

(μm). One such example is Klebsiella

pneumoniae, a Gram-negative encapsulated bac-

teria approximately 3 μm in length.

K. pneumoniae are phagocytosed and can survive

within macrophages in specialised vacuoles

called Klebsiella-containing vacuoles (KCV).

The size obviously poses difﬁculty visualising

bacteria, and when trying to unravel the complex

host–pathogen interactions, even more so.

In this section, we will describe techniques

utilised in microbiology research to analyse facets

of bacterial function and growth.

2.12 Spectrophotometry

Spectrophotometry is the use of light to measure

the turbidity of a solution. A calibration curve of

known bacterial number will correspond to a

given % Transmission of light, providing a mea-

surement tool that greatly increases the speed at

which bacterial number can be established. This

is used extensively in microbiology laboratories

to equilibrate bacterial numbers used in

experiments to ensure valid results. Using

K. pneumoniae as an example, at an optical den-

sity (OD) of 1, there are 5 ×10

bacterial cells/ml.

2.13 Bioscreen

This same principle can be used to determine

bacterial growth over time. The generation or

doubling time of a given bacterial species can

vary widely, again using K. pneumoniae as an

example, doubling time is approximately

20 min. Population growth consists of 4 phases:

lag phase, exponential phase, stationary phase

and decline phase. These phases can be deter-

mined by taking samples of bacterial cultures

every 20 min and measuring the optical density.

This process can be automated using a piece of

equipment that can read OD at speciﬁc time

intervals, the Bioscreen CTM Automated Micro-

bial Growth Analyzer (MTX Lab Systems,

Vienna, VA, USA) for example. This method

can be used to differentiate the ability of bacteria

to grow in different conditions. Figure 2.2 shows

the growth kinetics of K. pneumoniae in chemi-

cally deﬁned media supplemented with glucose,

and a strain of K. pneumoniae that has a meta-

bolic growth defect.

2.14 Plating

Another means of visualising population growth

of bacteria is counting of colony-forming units

(CFU) (Fig. 2.4a). Each CFU is assumed to have

grown from a single bacteria cell. In order to be

able to count the number, a sample is taken from a

bacterial culture every 20 min and serially

diluted. A given volume of the dilution is spotted

onto an LB agar (nutrient-rich, solid growth

medium) and spread evenly over the agar and

the bacteria are allowed to grow.

Bacterial species can be “visualised”using

selective agar. K. pneumoniae can metabolise

citrate. Simmons citrate agar’s sole nutrient

source is citrate and can therefore act as a selec-

tive media (Fig. 2.4a). Citrate can be metabolised

by many bacteria by myo-inositol is selective for

Klebsiella species. Citrate- myo-inositol agar is

used during gut colonisation infection

experiments with K. pneumoniae so only this

bacterium grows and the ability to colonise the

gut can be determined (Fig. 2.4b).

2.15 String Test

Infections caused by K. pneumoniae can be very

broadly divided into classical (cKp) and hypervir-

ulent (hvKp) sources, where cKP has been more

associated with hospital-acquired infections and

hvKp with community-acquired infections

(Wyres et al. 2020). Hypervirulent

K. pneumoniae was initially described from an

isolated strain in the mid-1980s in Taiwan (Cata-

lán-Nájera et al. 2017). The most characterised

virulence factors of K. pneumoniae are the cap-

sule, lipopolysaccharide, O-antigen and

siderophores as well as outer membrane proteins,

porins and Type1 and 3 ﬁmbriae (Lawlor et al.

2005; Paczosa and Mecsas 2016; Bruchmann

et al. 2021). Lipopolysaccharide (LPS) capsule

is one of the major constituents of the membrane

of Gram-negative bacteria and is typically com-

posed of an outer O-antigen, an oligosaccharide

core and inner lipid A molecule (Mills et al. 2017;

Bartholomew et al. 2019). One of the key viru-

lence factors for K. pneumoniae infection is the

characteristic capsule and, as previously

described, this can result in a hypermucoviscous

phenotype (Paczosa and Mecsas 2016). There are

at least 77 recognised capsular types, designated

as K1 or K2 for example, with at least 1345

distinct K-types that have been identiﬁed through

genome sequencing (Wyres and Holt 2016). As a

capsulated bacterium, K. pneumoniae appears

mucoid when grown on nutrient agar but the

hypermucoviscous designation is only given to

those strains that can produce an elongated ﬁla-

ment of ≥5 mm upon stretching with an inocula-

tion loop from an agar plate- the string test (Fang

et al. 2004).

2 Visualisation of Host–Pathogen Communication 33

Fig. 2.4 K. pneumoniae can be quantiﬁed by counting

colony-forming units (CFU). As there are too many when

concentrated, serial dilutions are performed. (a) Image of

1/10 serial dilution of K. pneumoniae 5×10

initial

suspension. At 10

-6

, the CFU can be counted (4) in

10 uL and the total number of CFU/mL calculated. (b)

The total CFU/mL can be calculated to determine the

survival of K. pneumoniae within macrophage by

quantifying the number of bacteria harvested from

macrophages at speciﬁc timepoints. (b) Representative

serial dilution from 10

-1

to 10

-4

of K. pneumoniae after

60 min contact with macrophages

The string test is essentially a very simple

technique whereby we can quantify the

hypermucoviscosity of a given bacterial culture.

A loop was used to lift a portion of a single colony

approximately 2 mm in diameter from a fresh LB

agar plate. The formation of viscous strings

>5 mm in length indicates a positive string test

(Fang et al. 2004).

2.16 Mass Spectrometry

Since the invention of modern mass

spectrometers in 1913 by electron discoverer

and Nobel Laureate in Physics Joseph John

Thomson (R. 1914), mass spectrometry has

become a valuable tool for biological research.

Mass spectrometry is an analytical technique used

to identify chemical components by measuring

mass-to-charge ratio of ions. The results of this

technique are presented as mass spectrums, a plot

of intensity as a function of mass-to-charge ratio.

These plots are used to determine the elemental or

isotopic nature, the masses of particles or

molecules within a given sample. Both pure and

complex mixtures, gaseous, solid or liquids can

be analysed in this procedure.

34 A. Dumigan et al.

Fig. 2.5 Schematic

diagram of a spectrometer

+ + + + ++ +

+ + + +

+ + + + ++

+ + + + +

ION SOURCE

ANALYZER

ABC

DETECTOR

RECORDER

Mass spectrometers have four main

components (Mellon 2003): an ion source, a

mass analyser, a detector and a recorder device

(Fig. 2.5). First, samples need to be ionised by the

ion source, with different techniques being used

and determining which kind of samples can be

analysed. One of the most used for the study of

biomolecules is matrix-assisted laser desorption/

ionisation (MALDI), coined for the ﬁrst time in

1985 (Karas et al. 1985). It consists of the use of a

pulse laser to irradiate the sample, which is mixed

with a suitable matrix material (DHB, sinapic

acid, ferulic acid, etc.) and applied to a metal

plate. The application of the laser triggers the

ablation and desorption of the sample, and its

ionisation by protonation or deprotonation, after

which it is accelerated into the analyser.

Another commonly used way to produce ions

is electrospray ionisation (ESI), where a high

voltage is applied to the sample, which is solved

in a mix of water, volatile organic compounds

(like methanol or acetonitrile) and some reagent

to increase conductivity (like acetic acid). This,

coupled with the use of a heated inert gas

(i.e. nitrogen) and the high temperature of the

ESI source, cause the nebulisation of charged

droplets that are ejected and accelerated into the

mass analyser (Bruins 1998).

After ionisation, sample’s ions are accelerated

into the mass analyser. Again, several analysers

can be used, but the most common ones for the

study of molecules of biological interest are

Time-of-ﬂight (TOF) mass analysers. They are

based on the fact that ions accelerated in an elec-

tric ﬁeld of constant and known voltage and

length (all englobed under a constant K, called

calibrating factor), the time they take to reach the

detector (t) is related to their mass-to-charge ratio

(m/z) according to the following equation

(El-Aneed et al. 2009):

z=Kt2

This way, the time that each of the ionised

atoms and molecules of the samples takes to

reach the detector is recorded. The most common

detector is an electron multiplier, which ampliﬁes

the signals received that is recorded and

represented most commonly in the form of a

mass spectrum (Fig. 2.6). This is usually com-

pared to a library of known mass spectra, which

allows the identiﬁcation of the compound present

in the sample.

2.17 Application in Microbiology

Mass spectrometry has become an important tool

in both microbiology research and clinical

diagnostics, being a fast and accurate method

that does not require specialised training and

being less expensive than other routine methods

(despite having an initial high cost in equipment)

(Singhal et al. 2015).

2 Visualisation of Host–Pathogen Communication 35

100

% intensity

100

1200 1400 1600 1800

1824

1840 1866

1840

1824

2000 2200

m/z 1200 1400 1600 1800 2000 2200

m/z

Fig. 2.6 Mass spectra obtained of lipid A extracted from Klebsiella pneumoniae strain Kp52145 grown in LB (left) and

recovered from lungs of infected C57BL/6 mice (right). Figures obtained from Llobet et al. (2015)

MALDI and ESI mass spectrometry has been

recently used for the rapid identiﬁcation and clas-

siﬁcation of bacteria and other microorganisms

(Sauer and Kliem 2010). Colonies from clinical

isolates can be analysed by MALDI-TOF to

obtain spectral ﬁngerprints that are compared to

those stored in databases provided by the mass

spectrometer supplier (Croxatto et al. 2012).

Also, direct identiﬁcation from urine or blood

samples can be performed, although this requires

some previous processing to concentrate the

microorganism, as well as eliminating other

compounds present in these ﬂuids that would

suppose a too complex mix to be analysed by

mass spectrometry.

It has also become a powerful proteomics tool

to study host–pathogen interactions (Sukumaran

et al. 2021). During an infection, mass spectrom-

etry can be used to analyse the protein composi-

tion of different compartments, as cell proteome

changes during Salmonella infection (Selkrig

et al. 2020), different proteome expression in

neutrophils from two mice strains infected with

Pseudomonas aeruginosa (Kugadas et al. 2019),

analysis of exosomes released from human

macrophages after Mycobacterium tuberculosis

infection (Diaz et al. 2016), or the

characterisation of post-translational modiﬁcation

after viral infections (Kulej et al. 2015). Also,

protein-protein interaction can be analysed by

the combination of afﬁnity puriﬁcation and mass

spectrometry (AP-MS) (Morris et al. 2014); this

has been recently used in the search of therapeutic

target against SARS-CoV-2 infection (Gordon

et al. 2020).

Not only proteins can be analysed in mass

cytometry. MALDI-TOF can be used for the anal-

ysis of nucleic acids, which has been applied to

the discovery of single-nucleotide

polymorphisms (Stanssens et al. 2004) and epide-

miological studies (Ecker et al. 2009).

Genetic analysis of environmental strains and

human isolates has distinguished four

phylogroups and determined that they should be

designated as distinct species: Klebsiella

pneumoniae (Kp1), Klebsiella quasipneumoniae

subspecies quasipneumoniae (Kp2), Klebsiella

quasipneumoniae subspecies similipneumoniae

(Kp4) and Klebsiella variicola (Kp3) (Brisse

and Verhoef 2001; Rosenblueth et al. 2004; Holt

et al. 2015). Recent taxonomic updates and

matrix-assisted laser desorption ionisation time

of ﬂight (MALDI-TOF) mass spectrometry

(MS) has further identiﬁed K. pneumoniae

phylogroups Kp5 and Kp6. Together these are

recognised as the K. pneumoniae complex

(KPC) (Rodrigues et al. 2018). Although all

KPC species can cause human infections,

K. pneumoniae is the strain most isolated from

patients (Holt et al. 2015).

Finally, mass spectrometry has been also used

to decipher the structure of bacterial lipopolysac-

charides. In Klebsiella pneumoniae, it has been

shown that it modiﬁes lipid A structure in a

tissue-dependent manner (Fig. 2.6) (Llobet et al.

2015). It has been also described how two

acyltransferases, LpxL1 and LpxL2, catalyse the

addition of laurate and myristate, respectively and

have an important role in Klebsiella virulence

(Mills et al. 2017).

36 A. Dumigan et al.

2.18 Closing Statement

In this chapter, we have given a brief overview of

the importance of the study of microorganisms

and of our own body’s defence against infection

via specialised cells of the immune system. It is

important to remember that we have evolved to

have a largely symbiotic relationship with

microorganisms in that we have “good bactieria”

in our guts which aid digestion. However, when

infected with pathogenic bacteria or with nor-

mally commensal bacteria in a compartment of

the body ill-adapted to the presence of speciﬁc

bacteria, the example we have provided is that of

the opportunistic pathogen Klebsiella

pneumoniea, we begin to see morbidity and mor-

tality. Therefore, the ﬁeld of host–pathogen com-

munication is the study of how bacteria and host

interact in order to understand the pathogenicity

of disease and develop novel effective treatments.

Visualisation of intricate cellular structure,

morphology and cell-to-cell interactions is an

essential aspect to the study of host–pathogen

interactions. Continued development of

visualisation techniques and advances in technol-

ogy available to biomedical researchers and

clinicians is integral for the continued progress

of medicine. We have covered the development

of microscopy and the use of standardised

staining and dye protocols which have been

around for over a century and are still used

today. One such stain is H&E used by

pathologists and researchers worldwide. The use

of FISH to visualise gene expression in situ. The

basis of ﬂow cytometry as a highly useful tool in

understanding immune cells and the development

of cutting-edge imaging f1ow cytometry and

mass spectrometry. The use of these technologies

in conjunction with standard protein immunoblot-

ting has helped researchers not only determine

cell activation and recruitment but also to delin-

eate cell signalling processes involved. The use of

plating techniques allows us to decipher bacterial

loads in vitro and in vitro. Also the use of simple

string tests and selective media to characterise

bacterial strains are all used in combination.

Indeed as researchers we depend on the versatility

of techniques available in order to investigate

biological questions; this is key as there is not

one perfect technique but rather a combination

must be used to fully elucidate mechanisms at

play. Clinicians also routinely utilise plating,

microscopy and ﬂow cytometry to diagnose

patients and develop effective treatment plans.

Combining proteomics, cell surface expression

and intracellular staining We know that K.

psubverts inﬂammation and via the use of the

techniques described herein, we can observe how

K.p inﬂuences innate immune cells to evade the

immune response and promote its own survival.

This is very important work as with continued rise

of AMR in several bacterial pathogens worldwide

(WHO 2014), understanding of host–pathogen

interactions is key to developing effective thera-

peutic interventions.

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A Review of Pathology and Analysis

of Approaches to Easing Kidney Disease

Impact: Host–Pathogen Communication

and Biomedical Visualization

Perspective

Advanced Microscopy and Visualization of Host-

Pathogen Communication

Kacper Pizon, Savita Hampal, Kamila Orzechowska,

and Shahid Nazir Muhammad

Abstract

Introduction: In addition to affecting the

upper respiratory tract, severe acute respiratory

syndrome-coronavirus (SARS-CoV) and

SARS-CoV-2) can target kidneys resulting in

disease impact. There is a lack of effective

treatment for SARs-CoV and SARS-CoV-2,

and so one approach could be to consider to

lower the probable risk and onset of disease

amongst immunocompromised and

immunosuppressed individuals and patients.

Angiotensin Converting Enzyme 2 (ACE2)

has a promising impact including acting

against SARs-CoV and SARS-CoV-

2 symptoms. Current literature states that

ACE2 is expressed across several physiologi-

cal systems, including the lungs,

cardiovascular, gut, kidneys, and central ner-

vous, and across endothelia. Aims: This chap-

ter seeks to investigate causes and potential

mechanisms during SARS infection (CoV-2),

renal interaction, and the effects of acute kid-

ney Injury (AKI). Objectives: This chapter

will provide an overview of microscopy and

visualization of host–pathogen communica-

tion and principles of ACE2 in the context of

immunology and impact on renal pathophysi-

ology. Design: This chapter focuses to provide

basic principles of ACE2 and the analysis and

effect of immunology and pathological

components important in relation to SARs

infection. Discussion: There has been a surge

in literature surrounding mechanisms

attributing to SARS-CoV and SARS-CoV-

2 action on immune response to pathogens.

There is an advantage to implementing ACE2

treatment to improve immune response against

infection. Conclusion: ACE2 may provide

appropriate strategies for the management of

symptoms that relate to SARS-CoV and

SARS-CoV-2 in most immunocompromised

or immunosuppressed patients. Visualization

of ACE2 action can be achieved through

microscopy to understand host–pathogen

communication.

K. Pizon · S. Hampal · K. Orzechowska

Department of Life Sciences, Coventry University,

Coventry, England, UK

The Renal Patient Support Group (RPSG), Coventry,

England, UK

S. N. Muhammad (✉)

Department of Health, and Life Sciences, Coventry

University, Coventry, England, UK

University Hospitals Bristol NHS Foundation Trust,

Bristol, England, UK

e-mail: ad9152@coventry.ac.uk

#The Author(s), under exclusive license to Springer Nature Switzerland AG 2023

E. Abdel Meguid et al. (eds.), Biomedical Visualisation, Advances in Experimental Medicine and

Biology 1406, https://doi.org/10.1007/978-3-031-26462-7_3

42 K. Pizon et al.

Keywords

Nephrology · Immunology · Virology ·

Visualization · Host–pathogen · Biomedical

sciences

3.1 Introduction: Discovery

of Coronaviruses

Since December 2019, the severe acute respira-

tory syndrome Coronavirus 2 (SARS-CoV-2)

spread worldwide causing COVID, to be prob-

lematic and implicating health risks across the

globe (Gheblawi et al. 2020). Coronaviruses can

affect multiple organs in humans, including

kidneys, increasing mortality, especially in those

who are immunosuppressed and/or immunocom-

promised. Moreover, the lack of treatment has

forced biomedical and pharmaceutical industries

to investigate and develop treatments to prevent

the exaceberation of COVID-19 in the general

population (Gheblawi et al. 2020). There is a

lack of effective treatment for SARS-CoV and

the more recent SARS-CoV-2, and so one

approach could be to consider to lower probable

risk and onset of disease amongst immunocom-

promised and immunosuppressed individuals and

patients (Gheblawi et al. 2020; Guo et al.

2020a,b). The research surrounding COVID-19

has been progressive; it is important to reduce

disease mortality at the epidemiological and pop-

ulation level. However, the understanding of the

structure, and pathology of Coronaviruses, espe-

cially surrounding host–pathogen communica-

tion, and visualization, requires investigation.

3.2 Aims and Objectives

This chapter seeks to investigate the causes and

potential mechanisms of severe acute respiratory

syndrome (SARS) infection (CoV-2), renal inter-

action, and the effects of acute kidney injury

(AKI). This chapter also provides a review of

the pathology and analysis of approaches to eas-

ing kidney disease impact by exploring host–

pathogen communication, providing a biomedical

visualization perspective, and highlighting the

principles of Angiotensin Converting Enzyme

2 (ACE2) in the context of immunology and

infection to understand the impact on kidney

disease.

3.3 Biomedical Classification

Coronaviruses are more recent discoveries than

adenoviruses; both fall under the same category,

but adenoviruses have more taxonomy

investigated (Wang et al. 2020; Beyerstedt et al.

2021). The ﬁrst Coronavirus strain (B814) was

discovered in 1965 by scientists Tyrrell and

Bynoe, where biomedical specimens were col-

lected from the respiratory tract of a patient with

a common cold (Mahase 2020). Thereafter,

Hamre and Procknow were then able to grow

isolated virus strains, including B814 and 229E,

which improved early biomedical visualization in

the context of extraordinary viruses (Mahase

2020).

Linked to early biomedical visualization of

common cold and COVID were isolates to virus

strains associated with avian bronchitis and mice

hepatitis; these were originally explored by

Almeida and Tyrrell in 1967 (Mahase 2020).

The same year at the National Institute of Health

in Bethesda, six additional viruses with morpho-

logical similarities were isolated and grown in

organ cultures, instead of the usual cell mono-

layer culture (Mahase 2020). In 1968, a more

in-depth investigation of viruses using electron

microscopy revealed comparable properties

between them, for example, size, lipid layer, and

the presence of surface spikes (i.e., protein),

which resemble similar structures to the Adeno-

virus (Lupala et al. 2022). Figure 3.1 provides an

overview of the progression and discovery of

novel subgroups of viruses/coronaviruses.

Based on taxonomy and pathological features,

a team of eight virologists decided to classify

them as Coronaviruses, due to their characteristic

appearance (Mahase 2020; Beyerstedt et al.

2021). Ongoing research and microscopy

advances surrounding Coronaviruses provided

greater knowledge of viral structure and function,

to discover therapeutic possibilities surrounding

unpredictable outbreaks. Advanced microscopy

and visualization are pertinent throughout scien-

tiﬁc practices and more so in exploring links to

COVID-19 virus and the pandemic (Song et al.

2019).

3 A Review of Pathology and Analysis of Approaches to Easing Kidney Disease... 43

Fig. 3.1 The progression and discovery of novel subgroup of viruses/coronaviruses. HCoV human coronavirus, MERS

middle east respiratory syndrome, SARS severe acute respiratory syndrome (Abdul-Fattah et al. 2021)

Regarding pharmaceutics, Angiotensin-

Converting Enzyme 2 (ACE2) has diverse inter-

play, as SARS-CoV-2 impounds respiratory

health and the implications prompt symptoms

that have been highlighted in the literature

relating to the COVID-19 pandemic. The

implications on health and disease have become

more obvious following the SARS outbreak in

Wuhan province, China in 2003 (Mahase 2020;

Gheblawi et al. 2020). In the next section, an

analysis of Coronaviruses in correlation with

renal health, and novel therapeutic strategies

relating to preventing cardiovascular disease

(CVD) and COVID-19 symptoms are considered.

44 K. Pizon et al.

3.4 The Renal Health

and COVID-19 Correlation

It has been highlighted that the ﬁrst pandemic

caused by Coronavirus SARS-CoV occurred in

2002–2003, in Asia, wherein 8096 patients

evaluated positive for the virus and as a result,

774 deaths had been documented (Sama et al.

2020). Despite common acute respiratory failure

caused by diffuse alveolar damage, multi-organ

failure was also noticeable wherein there were

cases of severe diarrhea, hepatic failure, and

Acute Kidney Injury (AKI). It was in 2005, an

investigatory team from Hong Kong, retrospec-

tively highlighted that 536 SARS cases (6.7%)

developed AKI (Chu et al. 2005). Moreover, the

mortality amongst this group was 91.7%, which is

signiﬁcantly higher than patients without any

symptoms of AKI, which is 8.8% (Chu et al.

2005).

Gheblawi et al. (2020) and Guo et al. (2020b)

also emphasized the importance of AKI in the

context of SARS and Middle East Respiratory

Coronavirus (MERS-CoV). MERS-CoV induced

two epidemics; one in 2012 surrounding

countries in the Middle East and one in 2015 in

South Korea (Gheblawi et al. 2020; Guo et al.

2020a). The MERS-CoV devastated the lives of

2562 people resulting in 881 deaths. To deter-

mine possible nephrological complications

associated with MERS-CoV, investigators from

South Korea examined 30 patients by monitoring

vital signs, bacterial culture of body ﬂuids and

virus through polymerase chain reaction

investigations. Within a testing group, proteinuria

occurred in 40% of cases, haematuria in 63.3%

and AKI developed in 26.7% of patients, mostly

in the elderly (Gheblawi et al. 2020; Guo et al.

2020b).

The current outbreak of SARS-CoV-2, which

originated from Wuhan province, China, spread

worldwide threatening life. Within the period of

September 1, 2020, to August 31, 2021,

5,642,190 cases of COVID-19 were detected in

England, wherein 94,815 cases led to death

(UKHSA 2022). Despite being uncommon,

renal complications have been correlated to

pathophysiology detrimented by the effects of

COVID-19 infection, mainly because of AKI

(Gheblawi et al. 2020). Moreover, the risk for

individuals who are prone to renal insufﬁciency

to contract COVID-19 is higher, and thus

interventions have been required to preclude fur-

ther renal complications and CVD (Huang et al.

2020). The next section provides an in-depth

overview of renal health and ACE2 at the micro-

scopic level.

3.5 Renal Cells and COVID-19

Ribonucleic Acid (RNA) sequencing database

assessment had demonstrated high expression of

ACE2 and transmembrane protease serine

2 (TMPRSS2) in proximal tubular cells and

podocytes, which might indicate a higher afﬁnity

for SARS-CoV2 (Murray et al. 2020). Micro-

scopically, the presence of ACE2 within proximal

tubular cells might indicate direct interaction with

Coronavirus, which subsequently reﬂects kidney

damage. However, it does not exclude the possi-

ble ischemic damage to the proximal tubular cells

from hemodynamic alterations, informing that the

knowledge surrounding direct interactions is

vague and unspeciﬁed (Yang et al. 2017; Henry

and Lippi 2020).

Immunobiology data indicate that lower

expression of ACE2 correlates with a severe

effect of disease owing to the downregulation of

ACE2, which leads to a reduced anti-

inﬂammatory effect (Murray et al. 2020). The

severity of the illness increases with the lower

expression of ACE2 receptors, which might

again contradict the correlation among a direct

interaction between SARS-CoV-2 and renal

cells (Yang et al. 2017; Henry and Lippi 2020).

Figure 3.2 provides an overview of ACE2 recep-

tor expression in human organs.

Several investigations present the theory of

SARS-CoV-2 tropism, which leads to viral infec-

tion of proximal tubular cells (Brake et al. 2020;

Yang et al. 2017; Henry and Lippi 2020). The

argument is based on the presence of viral

particles within urine detected from COVID-19

patients, especially within the second and third

week of infection, and when the shedding of

SARS-CoV-2 occurs (Guruprasad 2021b). How-

ever, histological examination negates the pres-

ence of active immune-mediated

glomerulonephritis due to the absence of common

indicators like electron-dense deposits and mild

proteinuria (Baral et al. 2020). Moreover, the

number of viral particles differs among several

ﬁndings due to the lack of standardized sample

collection and appropriate PCR assays conducted.

Data highlight either little or no detection, and

others present a high number of viral particles,

which leads to inconsistent data (V’kovski et al.

2021).

3 A Review of Pathology and Analysis of Approaches to Easing Kidney Disease... 45

Fig. 3.2 Provides an overview of ACE2 receptor expres-

sion in human organs. As the kidney presents a higher

level of ACE2 than the lungs, it leads to speculation of

possible direct infection of HCoV-SARS-19 within kidney

cells (Xu et al. 2020)

A novel infection route was identiﬁed for

SARS-CoV-2, which was the spike protein

attachment to transmembrane glycoprotein

CD147 (Guruprasad 2021a). As CD147 is

expressed in various cells all around the body,

including blood cells and proximal tubules of the

kidney, the possible viral entry might affect the

cell cycle, which affects inﬂammatory responses

and further development of several diseases

(Guruprasad 2021b). Due to the novelty of initial

data across the literature, the idea of direct infec-

tion within the renal cells is still speculative

(Murray et al. 2020; Wang et al. 2020). The

next section provides more depth surrounding

the analysis of COVID-19 in the context of tax-

onomy and genera, accordingly.

3.6 Biomedical Visualization

and Analysis of COVID-19

The Coronavirus is divided into four genera:

(1) Alpha-Coronavirus (HCoV-229E, HCoV-

NL63), (2) Beta-Coronavirus (HCoV-OC43,

HCoVHKU1, SARS-CoV, MERS-CoV,

SARS-CoV2), which originate from bats,

(3) Gamma-Coronavirus, and (4) Delta-

Coronavirus can be transmitted from types

of birds and swine gene pools (Dhama et al.

2021). A SARS-CoV-2 virion contains an unseg-

mented, single-stranded, positive-sense RNA

genome of around 30 kb, which encodes crucial

viral protein used to create progeny viruses and

the four essential structural proteins, called mem-

brane (M), envelope (E), nucleocapsid (N), and

spike (S) (V’kovski et al. 2021). As far as air-

borne viruses are concerned, SARS-CoV-2 is

transmitted between humans due to the entering

of viral particles into the upper respiratory tract

(Dhama et al. 2021). The viral entry occurs by the

binding of the spike glycoproteins, which are

homotrimers present on the surface of the Coro-

navirus to the ACE2 receptor (Guruprasad

2021a).

46 K. Pizon et al.

The spike proteins are cleaved by host

proteases into subunits: S1 subunit for receptor

binding and S2 subunit for membrane fusion

(Guruprasad 2021b). Microscopically, the

infected cells are forced to translate the proteins

required for generating the virus’s progeny

(Mehrbod et al. 2021). The next section provides

context surrounding the renin–angiotensin–aldo-

sterone system (RAAS) pathway and the role of

ACE2 during COVID-19.

3.7 The Cytokine Storm

Perspective

and Understanding Renin–

Angiotensin–Aldosterone

System (RAAS) Pathway

and Role of ACE2 During

COVID-19

The RAAS is a multi-hormonal regulator of blood

volume and systemic vascular resistance.

Angiotensinogen is cleaved by renin forming

inactive decapeptide Angiotensin I (Ang I),

which is next converted by angiotensin-

converting enzyme (ACE) into active octapeptide

Angiotensin II (Ang II). The binding of Ang II

with adequate angiotensin receptor (AT1 or AT2)

leads to hypertension, inﬂammation, and multi-

organ failure (Beyerstedt et al. 2021). Works by

Gheblawi et al. (2020), Guo et al. (2020a)

highlight common and potent antiviral drugs,

however, before these, ACE2 had been available

to prompt blood ﬂow between renal afferent and

efferent arterioles, thus oxygenating cells

throughout general physiology.

ACE2 performs a more protective role by

degrading Ang II to active peptide, Ang 1–7

through cleavage of the carboxy-terminal phenyl-

alanine in Ang II (Povlsen et al. 2020). Products

of ACE2 action binds with Mitochondrial Assem-

bly Protein-1 (MAP-1) receptor leading to vaso-

dilation, vaso-protection, and cardio-protection,

showing regulatory roles of both receptors

(Povlsen et al. 2020). However, ACE2 receptors

are inhibited during SARS-CoV-2, which

downregulates protective action and leads to

severe development of COVID-19 by hypercoa-

gulation and microangiopathy. The infected cells

are forced to translate proteins required for

generating the virus’s progeny, which also

provokes immune action (Povlsen et al. 2020;

Lupala et al. 2022) (Fig. 3.3).

Innate immune cells and macrophages are

involved to present viral antigens to T-Helper

(TH) cells, which release interleukin-12 to trigger

the TH1 cells (Povlsen et al. 2020). Subsequently,

TH1 stimulate B cells to produce antigen-speciﬁc

antibodies and T-Killer cells (CD8+ and TK) to

target cells containing viral antigen (Gheblawi

et al. 2020). Using the Nuclear Factor kappa-

light-chain-enhancer of activated B cells

(NF-kB) signaling pathway, Pro-T-cell activate

inﬂammatory cytokine production, secreting che-

mokine, and cytokines such as IL-8, TNF-α, IL-6,

IL-1β, CCL-2 (C-C Motif Chemokine Ligand 2),

-3, -5 (Sama et al. 2020; Song et al. 2019;

UKHSA 2022).

Accumulation of immune proteins creates a

cytokine storm, which leads to tissue damage,

resulting in multi-organ failure and death

(Gheblawi et al. 2020; Wang et al. 2020). The

limitations related to post-mortem examinations

of renal cells demonstrate evidence of AKI

(Gheblawi et al. 2020; Guo et al. 2020a,b). At

the same time, early COVID-19 cases have

presented involvement, mainly IL-6 which,

induces renal endothelial cells to prompt

pro-inﬂammatory cytokines (Gheblawi et al.

2020; Guo et al. 2020a,b; Wang et al. 2020).

3 A Review of Pathology and Analysis of Approaches to Easing Kidney Disease... 47

Fig. 3.3 The visualization of molecular action

undergoing differentiation during COVID-19 Infection.

HCoV-SARS binds to ACE2, which not only leads to its

entry but also irregular balance between Ang molecules,

resulting in the severity of the disease. ACE2 is also

pharmacologically related to Angiotensin Receptor

Blockers (ARBs), ARBs are inhibitors that dilate blood

vessels to treat conditions such as hypertension. Permis-

sion to use ﬁgure sought from Guo et al. (2020a)

Additionally, sepsis may be a factor in

COVID-19 patients, due to general physiology

being impacted. Within damaged organs, endo-

toxin overload leads to septic shock, which

induces AKI and renal damage (Henry and

Lippi 2020). Works by Beyerstedt et al. (2021),

Gheblawi et al. (2020), Guo et al. (2020a), Wang

et al. (2020) go into more microscopic detail

regarding ACE1/ACE2 gene polymorphism.

The next section provides more depth

surrounding immunobiology and the complement

system. The next section will consider

immunobiology and the complement system.

3.8 Immunobiology and The

Complement System

Upon recognition of SARS-CoV-2, complement

system becomes active and promotes pathogen

response via lectins and the alternative pathway.

The numerous anaphylatoxins, like C3a and C5a,

bind to speciﬁc receptors, leading to stimulation

of histamine, leukotrienes, and prostaglandins,

which results in ﬂushing, hypoxia, vasodilation,

and hypotension (Murray et al. 2020). Addition-

ally, an activated alternative pathway generates

C5b-9 in tubules apical brush border as a

response to the virus, leading to over-

accumulation and tubulointerstitial damage

(Henry and Lippi 2020).

The disruption of lymphocytes and role in the

maintenance of immune hemostasis leads to

lymphopenia, which has been observed in

patients with severe COVID-19 symptoms. As

those cells contain ACE2 receptors, SARS-CoV-

2 might target them, reducing the number of

CD4+ and CD8+ T Cells. This results in unequal

distribution of immune response, hyperactivation

of the innate immune system and a long process

of terminating the virus (Murray et al. 2020).

Another factor of lymphopenia might be induced

inﬂammatory responses, which prompts lympho-

cyte apoptosis, leading to lymphocyte ratio dis-

proportion (Beyerstedt et al. 2021). Through

analyses, patients with severe COVID-19 have

expressed injury around the spleen and lymph

nodes, which suggests that the virus can damage

lymphatic organs reducing contribution to

immune hemostasis (Mahase 2020).

48 K. Pizon et al.

There may be some overlap between immune

responses, after the detection of a foreign patho-

gen, facilitating hypercoagulability. Initially,

circulating pro-inﬂammatory chemicals activate

blood monocytes (Chowell et al. 2015). Endothe-

lial cells are activated by cytokines and virus

particles, which create adhesion molecules and

monocyte chemo-attractants, leading to the

recruitment of activated monocytes to endothelial

cells (Beyerstedt et al. 2021). Endothelial cells

also use neutrophils, which conduct coagulation

contact pathway, by realizing neutrophil extracel-

lular traps, which activate platelets. Additionally,

tissue factors might be exposed due to any endo-

thelial damage. The blood viscosity increases as

levels of oxygen decrease due to hypoxia induced

by COVID-19 leading to the stimulation of

thrombosis (Baral et al. 2020). Figure 3.4

provides an overview of the complement system’s

possible contribution to inﬂammatory response in

severe COVID-19.

Thrombosis laboratory indexes like partial

thromboplastin time, prothrombin time, and inter-

national normalized ratio are elevated, and

D-dimer, which correlates with high mortality

rate in COVID-19 patients (Yang et al. 2017).

Additionally, peripheral blood smears express

low ﬁbrinogen levels and thrombocytopenia

with schistocytes under morphology. Several

factors may prompt microthrombi and

microangiopathy, which elevate the risk of

micro-infarctions across cardiovascular,

respiratory, and renal physiological systems

inducing irreversible damage where COVID-19

symptoms are present (Yang et al. 2017).

Understanding rhabdomyolysis is also impor-

tant because it is caused by muscle breakdown

arising from numerous factors, such as overexer-

tion, trauma, toxic substances, or healthcare

complications over time. Autopsy data of

COVID-19 patients present acute proximal tubu-

lar damage and glomerular ﬁbrin thrombi with

ischaemic collapse (Henry and Lippi 2020).

There are several theories suggesting viral-

induced rhabdomyolysis. It might be induced by

a cytokine storm damaging muscles, circulating

toxins, muscle degeneration, or direct virus inva-

sion. Due to the novelty of COVID-19, there is

little transparency between a correlation

surrounding rhabdomyolysis and AKI in symp-

tomatic COVID-19 patients (Yang et al. 2017;

Henry and Lippi 2020). The next section provides

an overview of organ crosstalk and biomedical

communication.

3.9 Organ Crosstalk

and Biomedical

Communication

Linked to COVID-19 is acute respiratory distress

syndrome (ARDS). ARDS are life-threatening

disorders wherein the lungs cannot take in oxygen

for the body to maintain physiologically, leading

to incapacities surrounding crosstalk between

respiratory, cardiovascular, and renal systems

(Povlsen et al. 2020; Park and Faubel 2021). As

the COVID-19 pathology shares similar

symptoms to ARDS, and within both groups of

patients where AKI has been detected, it is

postulated that AKI develops from organ

crosstalk between the lung–kidney axis (Murray

et al. 2020).

Several factors, including an inﬂammatory/

immune response and the secretion of circulating

compounds, can interact with kidney cells and

cause harm to ARDS patients. The reduction of

oxygen transport within organs also contributes to

the development of AKI (Park and Faubel 2021).

A study in 2019 showed, that 70% of patients

3 A Review of Pathology and Analysis of Approaches to Easing Kidney Disease... 49

Fig. 3.4 The presentation of the complement system’s possible contribution to inﬂammatory response in severe COVID-19 (Risitano et al. 2020)

with healthy kidneys and ARDS developed AKI,

conﬁrming lung involvement in the development

of AKI, and suggesting comparable results for

COVID-19 infection (Park and Faubel 2021).

50 K. Pizon et al.

3.10 Current Treatments

and COVID-19

3.10.1 Reduction of Accessibility

of SARS-CoV2 to ACE2

Receptors

The severity of COVID-19 has led to an increased

focus on developing appropriate treatment against

SARS-CoV-2 to decrease the impact of the pan-

demic and protect the most vulnerable patients

(Florindo et al. 2020). The main aims of treatment

focus on blocking the virus from attaching itself

to the ACE2 receptor, which would stop the viral

entry (Ni et al. 2020). Knowledge about

receptors, action, and the reaction of each sub-

stance within the cells during SARS-COV-

2 infection would contribute to the development

of appropriate COVID-19 treatment (Ni et al.

2020).

One way to block the spread of SARS-COV-

2 is to introduce genetically modiﬁed ACE2,

called Human Recombinant Soluble (HRS)

ACE2, which would reduce the number of viral

attachments to the actual cells. The drug APN01

contains HRS ACE2 and is currently in Phase II

trial testing for lung disease (Abd El-Aziz et al.

2020). Previously, Arbidol 20 has been used in

treatment against inﬂuenza virus, which impairs

viral entry (Li et al. 2021). It works as a virus-host

cell fusion inhibitor and due to its broad-spectrum

antiviral application is currently clinically tested

against SARS-CoV-2 (Li et al. 2021).

A similar effect can be observed with the use

of a soluble form of ACE2 (Krishnamurthy et al.

2021). Soluble ACE2 is naturally found within

plasma (Yeung et al. 2021). Increasing its con-

centration in plasma would promote the attach-

ment of SARS-CoV-2, reducing the number of

pathogens entering the cells (Beyerstedt et al.

2021). This approach not only reduces severity

of disease but also preserves tissue ACE2 (Yeung

et al. 2021).

3.11 Anti-Virals on COVID-19

Antivirals, which are used for different viral

infections, are currently being tested for SARS-

Cov-19. As some of them have a broad range

application, this also allows them to be

investigated in suppressing the infection. Some

Antivirals include:

3.11.1 Lopinavir

After the SARS-CoV epidemic, the drug

Lopinavir was approved as an effective antiviral

due to its ability to inhibit viral action in in-vitro

environments (Tripathy et al. 2020). Lopinavir is

a protease inhibitor, which blocks protease’s

action, preventing the virus from creating prog-

eny within the cell. Originally, this treatment was

dedicated to HIV patients but due to a positive

decrease in mortality among patients with SARS-

CoV, it has been suggested that it might have

potential against SARS-CoV2 as well. Unfortu-

nately, there is no signiﬁcant decrease in the

mortality of COVID-19 patients (Yeung et al.

2021).

3.11.2 Hydroxychloroquine

Hydroxychloroquine is treatment with a safe pro-

ﬁle used for several ailments, mainly malaria and

due to anti-inﬂammatory properties for rheumatic

diseases (Ben-Zvi et al. 2011). This pharmaco-

therapy affects viral entry by changing the pH of

the vesicle, resulting in inhibition of several

enzymes, and ﬁnally disabling endocytosis, if it

is pH-dependent (Sinha and Balayla 2022). Addi-

tionally, glycosyltransferases are inhibited as

well, stopping post-translational modiﬁcations of

several viruses (Tripathy et al. 2020). Lack of

viral replication would lead to reduced immune

response, which would reduce the activation of

cytokine storm, and side effects of its action

(Ye et al. 2020).

3 A Review of Pathology and Analysis of Approaches to Easing Kidney Disease... 51

Theoretically, Hydroxychloroquine should be

an essential treatment for COVID-19 disease.

However, the examination of COVID-19 patients

treated with hydroxychloroquine leads to incon-

sistent data showing both a signiﬁcant decrease in

mortality among COVID-19 patients and little

effect in reducing mortality (Geleris et al. 2020).

Additionally, hydroxychloroquine is associated

with QT prolongation, which might cause

arrhythmia, so it is not an ideal treatment for a

patient who is taking other treatments for chronic

diseases, such as chronic kidney disease (CKD)

(Hooks et al. 2020).

3.11.3 Favipiravir

In China, a drug called Favipiravir is currently

used to treat severe inﬂuenza (Shiraki and

Daikoku 2020). It inhibits the action of the RNA

polymerase enzyme, which disables transcription

of the viral genome. This results in the prevention

of translating essential viral proteins

(Hassanipour et al. 2021). Preliminary data

obtained from the examination of Favipiravir

among patients with COVID-19 provided a

promising solution for the current pandemic

(Hassanipour et al. 2021). However, a review of

over 2700 studies concluded that there was no

evidence of reduced mortality among patients

with COVID-19 (Özlüşen et al. 2021).

3.11.4 Remdesivir

Remdesivir, developed by Gilead Sciences, was

developed to treat RNA-based viruses, which are

most likely to induce a global pandemic, such as

the Ebola virus, MERS, and SARS (Eastman

et al. 2020). The activity against SARS and

MERS led to suggest Remdesivir as a potential

candidate for COVID-19 due to the similarity

between those viruses (Eastman et al. 2020). It

is a mutagenic nucleoside, which terminates

pre-mature viral RNA (Snell 2001).

Within the presence of Remdesivir, a poliovi-

rus was constantly mutating to create a defence

mechanism against it (Crotty et al. 2001). Contin-

ually, the virus exposure to a high concentration

of Remdesivir can lead to a 9.7-fold increase in

mutations, which results in a genetic error of the

virus and its infectivity reduced by 99.3% (Crotty

et al. 2001).

Additionally, Remdesivir affects the DNA

synthesis process by reducing the reactive

hydroxyl group at the end chain terminators

(Mitsuya et al. 1990). Figure 3.5 provides an

overview of current treatments and COVID-19,

and Fig. 3.6 provides a speciﬁc overview

surrounding the inhibition of viral genome repli-

cation by Remdesivir.

Preliminary data presented informs that

patients treated with Remdesivir recover quicker

than those treated with a placebo (Beigel et al.

2020). It was also observed that Remdesivir

improved patients’well-being during the infec-

tion comparing them to a group treated with a

placebo (Beigel et al. 2020). Thanks to those

results, the Food and Drug Administration

(FDA) permitted the use of Remdesivir against

COVID-19. Despite positive outcomes of

Remdesivir usage, the mortality among patients

treated with it is still high, and the drug alone is

Fig. 3.5 An overview of Current Treatments and

COVID-19. The standard mode of action, in which

SARS-CoV-2 attaches to the membrane’s ACE2 and

enters the cell is presented on left. In the presence of

soluble ACE2, SARS-CoV-2 attaches to those units,

resulting in no membrane fusion (Kuba et al. 2021)

not sufﬁcient in curing COVID-19 disease

(Beigel et al. 2020).

52 K. Pizon et al.

Fig. 3.6 Inhibition of viral genome replication by Remdesivir (Al-Tannak et al. 2020)

3.12 Anti-Inflammatory Agents

on COVID-19 Recipient

As mentioned before, many factors might induce

organ dysfunction syndrome. Circulating cytoki-

nesis storm, which might be induced by

superimposed septic syndrome or directly by a

virus, causes severe damage to the body (Beigel

et al. 2020). To ease organ damage, the usage of

anti-inﬂammatory drugs was suggested to ame-

liorate COVID-19 infections (Beigel et al. 2020).

3.12.1 Steroids

During the SARS-CoV outbreak in 2002–2003,

the use of steroids was restricted due to the lack of

beneﬁcial results, and possibly harmful side

effects like delayed viral clearance, avascular

necrosis, diabetes, and psychosis (Russell et al.

2020). Corticosteroids were observed to increase

mortality among SARS patients, which indicates

the danger of using steroids to ease COVID-19

symptoms (Russell et al. 2020).

3.12.2 JAK-STAT Inhibitors

The JAK/STAT signaling pathway is present

within various cells and has been associated

with various roles in immune responses like

IL-2, IL-4, IL-6, IL-12 signaling, TH (T-Helper)

1, TH2, Treg cell and Th9, Th 17 cell differentia-

tion, the proliferation of viral selective CD8+ T

cells and B cell lymphoma (Satarker et al. 2020).

The presence of cytokine storm is essential for

dealing with a viral infection, however as men-

tioned before, cytokine overload might induce

organ damage or severe conditions like ARDS

(Luo et al. 2020). A solution has been suggested

that JAK-STAT inhibitors might decrease the

level of the pro-inﬂammatory cytokine, resulting

in general improvement of health among COVID-

19 patients (Luo et al. 2021). However, a lack of

an appropriate titer of cytokines might lead to

compromised immune responses and prolifera-

tion of SARS-CoV-2. Currently, studies of

JAK-STAT inhibitor medication called

Baricitinib, provide promising data, showing a

reduction in the number of deaths among

COVID-19 patients (Mahase 2022).

3 A Review of Pathology and Analysis of Approaches to Easing Kidney Disease... 53

While it is known that COVID-19 impacts

renal function, leading to increased mortality,

cytokine response of renal epithelium has not

been studied in detail. One team reported on the

genetic programs that activate human primary

proximal tubule (HPPT) cells by interferons and

suppression by Ruxolitinib, a Janus kinase (JAK)

inhibitor used in COVID-19 treatment

(Jankowski et al. 2021). Integration of future

research linked to AKI and COVID-19, as well

as other tissues, permit the avenue of kidney-

speciﬁc interferon responses. Additionally, new

drugs such as Ruxolitinib (dACE2) are being

trialed; recently discovered isoform (dACE2)

informs that initial data show promising results

linked to HPPT cells of the kidneys aligned to

ACE2, the SARS-CoV-2 receptor (Jankowski

et al. 2021).

3.12.3 Mesenchymal Stem Cell

Therapy

Cell-based therapy, particularly stem cell therapy,

has emerged as a potential therapeutic, with many

seeing prospects to heal incurable illnesses

(Lopes-Pacheco et al. 2019). The research

surrounding stem cells continues to generate

intrigue due to the potential including a high

proliferation rate, low invasive procedure, and

are free of ethical issues (Turner et al. 2021).

Among COVID-19 patients, stem cells focus on

modifying immune cell function, immune

responses and reducing inﬂammation-induced

lung injury (Golchin et al. 2019). Those stem

cells also produce growth factors, which promote

cell proliferation and prevent apoptosis (Adas

et al. 2021). Figure 3.7 provides an overview

surrounding visualization and mode of action

during mesenchymal stem cell therapy in

COVID-19 patients.

The decrease in mortality has been observed in

several studies, which focused on the usage of

Mesenchymal Stromal (stem) Cell (MSC) therapy

among COVID-19 patients (Metcalfe 2022).

Additionally, mesenchymal stem cells

signiﬁcantly shorten the time to clinical symptom

improvement in COVID-19 patients (Lanzoni

et al. 2021). Several clinical trials have shown

that MSCs are effective and safe for the treatment

of COVID-19 patients, particularly critically ill

patients, reducing clinical symptoms, hospital

stay, cytokine release, and mortality (Zumla

et al. 2020).

3.13 Conclusion

There has been a surge in the literature

surrounding mechanisms attributing to SARS-

CoV and SARS-CoV-2 action on the immune

response to a pathogen. SARS-CoV-2 patients

present an increased level of blood coagulation

factors and blood clotting complexities; the main

mediators include thrombin, tissue factor, and

ﬁbrinogen (Beyerstedt et al. 2021). SARS-CoV-

2 infection impairs the function of vascular endo-

thelial function, resulting in increased production

of thrombin and reduced ﬁbrinolysin, which

impacts clotting ability (Gheblawi et al. 2020).

While it is known that COVID-19 impacts kidney

function, leading to increased mortality, research

surrounding interrogation of cytokine response

on renal epithelium has not been substantiated.

3.14 Summary

There is an advantage to implementing ACE2

treatment to improve immune response against

infection; ACE2 may also offer appropriate

strategies for the management of symptoms that

relate to SARS-CoV and SARS-CoV-2 in most

immunocompromised or immunosuppressed

patients (Murray et al. 2020). However, what is

still not known is whether ACE2 should be

assigned at a population level to totally prevent

COVID-19 symptoms. That notwithstanding,

advances in laboratory practice, allow the imple-

mentation of pathology assays and techniques

including microscopy advances for visualization

and analysis to understand host–pathogen com-

munication to preclude kidney disease impact

(Gheblawi et al. 2020; Beyerstedt et al. 2021).

54 K. Pizon et al.

Fig. 3.7 Overview surrounding visualization and mode of action during mesenchymal stem cell therapy in COVID-19

patients (Câmara et al. 2021)

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Part II

Radiology and Patient Care

The Evolution of Equipment

and Technology for Visualising

the Larynx and Airway

Duncan King and Alison Blair

Abstract

Laryngoscopy and endotracheal intubation are

the core skills of an anaesthetist. The tools and

equipment used today are unrecognisable from

the methods used in the ﬁrst recorded attempts

at laryngoscopy over 200 years ago. The evo-

lution of the modern-day laryngoscopes has

mirrored advancements in technology within

general society, and particularly with regard to

computer and ﬁbreoptic technology over the

last 30 years. The development of these mod-

ern visualisation devices would not have been

possible without those that went before it, as

each new device has been inﬂuenced by the

previous. Video laryngoscopes have quickly

gained popularity as the primary intubating

device in many scenarios, driven by ease of

use as well as positive patient outcomes. While

it is still debated whether videolaryngoscopes

can replace direct laryngoscopy for routine

intubations, their effectiveness in difﬁcult

airways is unquestioned. This chapter will

cover the anatomy of the airway and the devel-

opment of technology from the rudimentary

creations in the early 1700s to the modern

larynsgocopes created in the twenty-second

century which allow the user to view the air-

way in more detail in order to secure endotra-

cheal intubation even in an airway where

intubation would be difﬁcult.

D. King

Northern Irish Medical and Dental Agency, Belfast,

Northern Ireland

A. Blair (✉)

Craigavon Hospital, Southern Health and Social Care

Trust, Craigavon, UK

e-mail: alison.blair@southerntrust.hscni.net

#The Author(s), under exclusive license to Springer Nature Switzerland AG 2023

E. Abdel Meguid et al. (eds.), Biomedical Visualisation, Advances in Experimental Medicine and

Biology 1406, https://doi.org/10.1007/978-3-031-26462-7_4

Keywords

Airway · Intubation · Larynx · Laryngoscope ·

Laryngoscopy

4.1 Airway

The larynx is located in the anterior aspect of the

neck at the level of the third to sixth cervical

vertebral bodies and lies at the junction between

the oesophagus and trachea. It is bordered by the

hyoid bone superiorly and the cricoid cartilage

inferiorly (Strandring et al. 2015). The primary

function of the larynx is to protect the trachea

from the aspiration of substances, by closing, in

a sphincter valve-like mechanism, upon mechan-

ical stimulation, typically during swallowing of

food or liquid. The larynx also plays a crucial role

in phonation and the regulation of breathing

(Pohunek 2004; Isaacs and Sykes 2002).

The anatomy of the larynx can be described by

its external anatomy and its internal anatomy.

It is composed of a rigid cartilaginous skele-

ton, and an inner lining of muscles. The skeleton

consists of three large unpaired cartilages (epi-

glottis, thyroid, and cricoid) and three smaller

paired sets of cartilages (arytenoids, corniculate,

cuneiform) (Strandring et al. 2015).

62 D. King and A. Blair

Epiglottis It is a leaf-shaped cartilage that

ﬂattens and moves down to cover the superior

opening of the larynx, preventing aspiration dur-

ing swallowing. It is attached by a “stalk”to the

anterior aspect of the thyroid cartilage. A clini-

cally important pair of pouch-like areas situated

between the epiglottis and the base of the tongue

are known as the valleculae. A Macintosh-style

laryngoscope is placed into this space to allow a

good view of the laryngeal inlet while intubating.

Thyroid Cartilage It is the biggest of the

cartilages of the larynx. It is composed of two

lamina and the anterior conjoint region between

the two is known as the laryngeal prominence

(colloquially known as the Adams apple)

(Strandring et al. 2015). Superiorly, the lamina

project to form superior horns, which attach to the

hyoid bone via the thyrohyoid membrane. Inferi-

orly the lamina forms inferior horns which attach

to the cricoid cartilage via the cricothyroid mem-

brane (Isaacs and Sykes 2002).

Cricoid Cartilage It is a signet ring-shaped car-

tilage that encircles the airway. The cricoid ring

consists of a tall posterior sheet or lamina and a

much narrower anterior arch. The cricoid carti-

lage itself signiﬁes the inferior border of the lar-

ynx and is found at the level of sixth cervical

vertebra. It articulates with the thyroid cartilage

as well as the arytenoids (Pohunek 2004). The

cricoid is the only laryngeal cartilage that is a

complete ring, a fact that is utilised clinically

during emergency intubation or non-fasted

patients. External pressure anteriorly over the cri-

coid cartilage causes compression of the oesoph-

agus and theoretically reduces the risk of

regurgitation and aspiration of stomach contents

(Roberts et al. 1994).

The paired arytenoids cartilages are three-

sided pyramidal shaped and they articulate with

the border of the lamina of the cricoid. The apex

of the arytenoids is where the corniculate

cartilages articulate. The lateral extension of the

arytenoids is known as the muscular process and

serves as an attachment point for the

cricoarytenoid muscles. The medial aspect of the

base is known as the vocal process. The vocal

ligament (part of the vocal cord) attaches at the

vocal process and extends across to the thyroid

cartilage (Strandring et al. 2015).

The epiglottis is attached to the arytenoids by

the aryepiglottic ligament.

The cuniform and corniculate cartilages are

embedded within these vocal folds. The

corniculate cartilages attach to the apex of the

arytenoids. They serve to reinforce and support

the aryepiglottic folds.

There are several membranes and ligaments of

the larynx that serve as support for the cartilagi-

nous skeleton. These can be described as extrinsic

if they attach the larynx to surrounding structures,

or intrinsic, when they connect the various laryn-

geal cartilages together and hold it as one single

functioning unit (Strandring et al. 2015).

4.1.1 Extrinsic

•Thyrohyoid membrane—connects the thyroid

cartilage to the hyoid bone (which is not tech-

nically part of the larynx)

•Hyoepiglottic ligament—attaches the hyoid

bone to the epiglottis

•Cricotracheal ligament—attaches the cricoid

cartilage to the ﬁrst tracheal ring.

4.1.2 Intrinsic

•Cricothyroid membrane—connects the cricoid

and thyroid cartilages. It is composed of two

parts; a single thickened median cricothyroid

ligament and two lateral cricothyroid

ligaments (also known as conus elasticus).

The cricothyroid membrane is clinically

important as the site of insertion of an emer-

gency airway in a “Can’t Intubate Can’t Ven-

tilate”scenario (Roberts et al. 1994).

•Quadrangular membrane—a relatively large

membrane that stretches between the lateral

aspect of the epiglottis and the arytenoid

4 The Evolution of Equipment and Technology for Visualising the Larynx and Airway 63

cartilages. The lower margin is thickened to

form the vestibular ligament, while the upper

margin forms the aryepiglottic folds

(Strandring et al. 2015).

The laryngeal cavity is the internal space between

the laryngeal cartilages and extends from just

below the epiglottis to the base of the cricoid

cartilage. When viewed during laryngoscopy

from above, there are two paired protrusions of

soft tissue into the laryngeal cavity. The superior

vestibular folds (or false vocal cords), and inferior

to that the white vocal folds (or true vocal cords).

The two-dimensional space in between the true

vocal cords is known as the rima glottidis.

4.1.3 Laryngeal Muscles

The complex range of functions performed by the

larynx is made possible by numerous sets of

muscles. These again can be classiﬁed into intrin-

sic (which are involved in phonation) and extrin-

sic muscles (which move the whole larynx

superiorly and inferiorly) (Roberts et al. 1994).

Extrinsic Muscles are compromised of the

suprahyoid groups (which generally elevate the

larynx) and the infrahyoid groups (which gener-

ally lower the larynx).

The Intrinsic Muscles together have three

distinct functions

•To close the vocal cords and inlet during

swallowing

•To open and relax the vocal cords during

inspiration

•To tense the vocal cords during phonation

The cricothyroid muscle acts to tense and

stretch the vocal cords and helps to create forceful

speech. The thyroarytenoid muscle relaxes the

vocal cords. The posterior cricoarytenoids abduct

the vocal cords, while the lateral cricoarytenoids

and the transverse arytenoid muscles adduct the

arytenoids, closing the glottis. The oblique

arytenoids and the aryepiglottic muscles adduct

the arytenoids, closing the glottis (Pohunek 2004;

Roberts et al. 1994).

4.1.4 Innervation of the Larynx

Laryngeal innervation is provided by two nerves,

the recurrent laryngeal nerve and branches of the

superior laryngeal nerve. Both are themselves

branches of the vagus nerve. The superior laryn-

geal nerve has two important branches—the inter-

nal and the external branches. The internal

receives sensory information from the glottis,

the supraglottis and the inferior aspect of the

epiglottis. The external branch provides motor

innervation to the cricothyroid muscle only.

The recurrent laryngeal nerve has sensory and

motor function. It receives sensory information

from the subglottis and provides motor innerva-

tion to all of the other intrinsic muscles of

the larynx. Of important clinical note, the

glossopharyngeal nerve provides sensory inner-

vation to the tongue base and the vallecula. It is

this that is stimulated during the process of laryn-

goscopy and endotracheal intubation (Strandring

et al. 2015).

4.2 Endotracheal Intubation

Endotracheal intubation is the process of inserting

a tube through the vocal cords and into the tra-

chea. It is a common, and essential skill in anaes-

thesia and done to protect and control the

patient’s airway (Collins 2014). There are in

effect an immeasurable number of indications

for endotracheal intubation. These indications

can be categorised into:

1. Protection of regurgitation of stomach

contents—e.g. in patients with low conscious

level.

2. Allow for mechanical ventilation during sur-

gery where spontaneous ventilation is not pos-

sible or adequate such as when muscle

relaxation is required.

3. Respiratory failure—due to any cause, such as

low conscious level, severe infection or mus-

cle weakness.

4. Partial or complete airway obstruction—which

again can be due to any cause (low conscious

level, infections, tumours).

64 D. King and A. Blair

5. To aid safe diagnostic procedures or

management—e.g. in patients who are intoxi-

cated or poorly co-operative, putting them-

selves or others at risk.

6. Shock syndrome of any cause—to allow tight

control of gas exchange in ICU (Hagberg

2018).

Controlling a patient’s airway is required in

the case of airway obstruction, often because the

patient has a low conscious level (through induc-

tion of anaesthesia, or other causes), resulting in

tissues around the upper airway losing their tone

and obstructing. Endotracheal tubes, when placed

within the trachea, result in a sealed circuit once

connected to a ventilator. This is achieved using a

balloon “cuff”in the distal aspect of the tube.

Inﬂating this cuff with air when placed in the

trachea creates this seal, therefore protecting the

airway from aspiration and allowing for positive

pressure ventilation (Hagberg 2018).

4.2.1 Laryngoscopy

Laryngoscopy is the broad term used for

visualisation of the larynx. It is a key practical

skill involved in anaesthetics, as it allows for

insertion of endotracheal tubes and is one of the

earliest key skills developed in anaesthetic

training.

Historically, humans have been obsessed with

developing means to visualise the internal body

structures, and the larynx is no different.

Physicians interest in visualising the larynx can

be traced to as early as mid-1700s (Burke 2004).

However, the historical credit for whom created

the laryngoscope as we know it is debated and

serves its roots in some related inventions from

the 1800s.

4.2.2 Early Devices

Despite the laryngoscope being the key tool of the

modern-day anaesthetist, early devices were in

fact created and used by surgeons. These early

creations were fairly rudimentary, and alterations

and adaptions have been made over the years by

successive scientists and resulting in the laryngo-

scope we know today (Burke 2004). In 1743 a

French surgeon named Leveret documented using

a highly polished metal spatula to visualise the

nasopharynx. He combined this with a snare-like

device to remove nasal polyps.

In 1807 German Physician Philipp von

Bozzini published a report on his device he called

his “light conductor”or “Lichtleiter”(Roth

2008). He described his device as “a simple appa-

ratus for the illumination of the internal cavities

and spaces in the living animal body”(Roth

2008). Von Bozzini’s device comprised a specu-

lum with a set of parallel tubes inside it. He

utilised light from a candle which is placed in

one of the tubes. This light is reﬂected down the

tube into the viewed body cavity, and the second

metal tube is used for viewing. This use of an

external light source differentiated his device

from earlier reﬂective devices. While von Bozzini

described using his device to visualise the naso-

pharynx and hypopharynx (among other

cavities), he never described visualisation of the

larynx (Bailey 1996; Pieters et al. 2015).

Benjamin Guy Babington in 1829 published a

report to the Hunterian society on his invention

the “glottoscope”which he used to view the lar-

ynx (Roth 2008). The “glottoscope”used sunlight

for illumination and consisted of a system of

mirrors as well as a separate tongue depressor

which he successfully used to visualise the larynx

(Pieters et al. 2015). The patient would be sitting

with their back to the sun and the user would then

hold a hand mirror, reﬂecting the sun’s light to the

back of the patient’s throat. A small mirror, which

was attached to a spatula, was then used to visu-

alise the larynx. Although there was documenta-

tion of Babington being able to view the larynx,

there was no reference made to the vocal cords

and their function (Burke 2004).

Despite all these earlier examples and

inventions, it is Manuel García who is generally

credited with the rather dubious title of “father of

Laryngology”. It is he who is considered to have

viewed the functioning glottis and vocal cords in

their entirety. García was a singer by trade, and in

a quest to improve his singing and teaching, he

wanted to learn how the vocal cords functioned.

Again, using the sun as an external light source,

García’s device used two mirrors, and was able to

observe his own functioning vocal cords and the

upper segments of his trachea (Burke 2004). In

his paper in 1855, García described the action of

the vocal cords during inspiration and

vocalisation and production of sound in the lar-

ynx (Bailey 1996).

4 The Evolution of Equipment and Technology for Visualising the Larynx and Airway 65

Laryngoscopy in this period was greatly hin-

dered by the requirement of an external light

source. This was restricted to using the sun, or a

candle, both of which were extremely impractical.

It was not until Thomas Edison invented the

lightbulb in 1878 that signiﬁcant advances could

be made.

4.2.3 From Indirect to Direct

All devices and methods used for visualising the

larynx and vocal cords had been under indirect

vision, i.e. through reﬂection using mirrors, that is

until Alfred Kristein ﬁrst described his device the

“Autoscope”in 1895 (Hirsch et al. 1986). His

device consisted of an electrical light source

termed the “electroscope”, which formed the han-

dle of the Autoscope. The light rays were focused

by a lens, and then reﬂected through 90 degrees

by a prism. A thick, rounded blade was attached

to the bottom of the handle, through which the

light was transmitted. The blade was used to

displace the tongue and epiglottis anteriorly,

therefore allowing direct visualisation of the

laryngeal inlet. Further developments by Kristein

produced two distinct blades for his Autoscope.

One which was placed into the vallecula, with

anterior pressure elevating the epiglottis allowing

visualisation of the larynx. The other, known as

the “intralaryngeal blade”, was placed beneath the

laryngeal surface of the epiglottis (Fig. 4.1). For-

ward and upward pressure lifted the epiglottis,

revealing the larynx below (Hirsch et al. 1986).

These two blades are clear predecessors to the

Macintosh and Miller blades. Kristein not only

pioneered the predecessor to the modern-day

direct laryngoscope, but he also understood the

importance of patient positioning for successful

laryngoscopy. He described “the body must be

placed in such a position that an imaginary con-

tinuation of the laryngotracheal tube would fall

within the opening of the mouth”(Hirsch et al.

1986). He understood that extension of the

atlanto-occipital joint is required, in what is now

colloquially termed the “snifﬁng the morning air”

position (Pieters et al. 2015).

Fig. 4.1 Kirstein’s modiﬁed autoscope. The standard

blade is shown attached to the handle, with the

intralaryngeal blade shown below. Medical Historical

Library, Harvey Cushing/John Hay Whitney Medical

Library. Reprinted with permission

Chevalier Jackson was the ﬁrst to combine

direct laryngoscopy with endotracheal intubation.

He invented a U-shaped laryngoscope with a

handle and a blade with an incorporated distal

light (Fig. 4.2). This differentiated his device

from Kristein’s earlier one, which used a proxi-

mal light source. Jackson’s laryngoscope also

incorporated a sliding base on the blade to allow

easy passage of an endotracheal tube (Moon et al.

2021). He was also the ﬁrst to introduce laryngos-

copy in the supine position, rather than the sitting

position that all previous methods had utilised.

Jackson also further emphasised the importance

of c-spine ﬂexion with head extension for laryn-

goscopy (Pieters et al. 2015).

Up until this time, the laryngoscope (or earlier

devices), were tools used by surgeons, primarily

for diagnostic purposes. It was clear that direct

laryngoscopy showed potential beneﬁts for

anaesthesia, and it was an American anaesthetist,

Henry Janeway, who was inﬂuential in

popularising the use of direct laryngoscopy in

anaesthetics. Surgery at the time was primarily

performed using inhalation anaesthesia, by plac-

ing a cone over the patient’s mouth and nose.

There was no protection against collapse of the

patient’s upper airway and obstruction by the

tongue, and there was also a high risk of aspira-

tion of blood, mucus, or vomit during surgery.

Janeway recognised these risks, and the potential

for tracheal intubation to alleviate these and

showed improved success of these surgeries by

achieving this (Burke 2004). He noted the difﬁ-

culty in achieving reliable and safe tracheal

intubation, and so developed a laryngoscope

with the sole purpose of aiding this (Pieters

et al. 2015). His device, known as the “specu-

lum”, incorporated a battery-powered distal light

source situated within the handle itself allowing

more manoeuvrability of the device. He also

added a central notch to the blade to guide the

tube during placement, as well as a slight curva-

ture to the distal tip to help direct the tip through

the vocal cords (Burke 2004).

66 D. King and A. Blair

Fig. 4.2 Jackson’s laryngoscope. Wood library-Museum

of Anesthesiology Reprinted with permission

The horriﬁcally disﬁguring injuries sustained

in World War 1, resulted in further advancements

within the ﬁeld of airway management in

anaesthetics. Two British Anaesthetists, Sir

Ivan W. Macgill and Edward S. Rowbotham,

described anaesthesia for reconstructive facial

surgery for British soldiers. They realised that

surgery for these soldiers was easier with the

airway secured with an endotracheal tube. From

the early 1920s, they described several alterations

and advancements to laryngoscopy, each of

which aimed at providing safe means to secure

the patients airway for facial surgery (Magill

1930; Rowbotham and Magill 1921).

Rowbotham and Magill (1921) described

using a laryngoscope developed similar to

Janeway’s earlier device, whilst incorporating a

“guiding rod”. This was a metal rod used to grasp

the end of the endotracheal tube and direct it into

the trachea under direct vision (Rowbotham

1920). Magill designed a U-shaped laryngoscope

in 1926, which itself was a modiﬁcation of the

earlier Jackson laryngoscope (Magill 1926).

Magill also altered his laryngoscope to be

“folding”—similar to a modern direct laryngo-

scope device. He suggested an intubation tech-

nique of inserting the blade in a “paraglossal

approach”, i.e. inserting the blade at the side of

the mouth rather than the midline, which helped

improve the view. He also put batteries into the

handle, continuing from Janeway’s earlier

advancement. Magill also introduced his now

ubiquitous “Magill forceps”, heavily based on

Rowbotham’s“guiding rod”, which help to direct

the tube into the trachea during endotracheal intu-

bation (Magill 1930).

A wide range of ultimately very similar laryn-

goscopy blades were being described at this time.

Robert Miller described his “straight”laryngos-

copy blade, (the so-called “Miller Blade”) in 1941

(Pieters et al. 2015; Miller 1941). While so-called

“straight”, there was still a slight curve to the

blade two inches from the tip. The blade was

designed to be placed under the epiglottis and

directly lift it onto the anterior wall of the larynx,

allowing a view of the vocal cords. In 1946 Miller

produced an infant version, which proved

extremely successful, and is still used today.

Infant anatomy is still considered by many to be

more suited to the use of a straight blade when

performing laryngoscopy.

Probably the most signiﬁcant and enduring

advancement in the ﬁeld of direct laryngoscopy

was made by Robert Macintosh. He described the

endotracheal intubation prior to the advent of

muscle relaxants as a “tour de force”, and

believed that the hallmark of a successful anaes-

thetist was “the ability to pass an endotracheal

tube under direct vision”(Scott 2009; Macintosh

1943). The majority of anaesthetists at the time

would often struggle to expose the vocal cords of

their unparalysed patients. The ground-breaking

1942 paper on the ﬁrst use of muscle relaxation

(Grifﬁth and Johnson 1942) was yet to be

published, and it was necessary to deepen the

patient with large doses of general anaesthesia

before attempting intubation (Scott 2009). The

invention of muscle relaxation allowed much eas-

ier exposure of the vocal cords, as well as easier

passing of endotracheal tubes through relaxed

cords. It also signiﬁcantly reduced the rate of

laryngospasm, the potentially life-threatening

condition where the vocal cords spasm and

clamp shut, preventing any air getting into the

lungs (Hagberg 2018).

4 The Evolution of Equipment and Technology for Visualising the Larynx and Airway 67

Macintosh felt that many anaesthetists were

struggling to consistently intubate patients due

to issues with poor technique. During direct lar-

yngoscopy, the standard practice at the time

involved passing a straight blade in the midline

and lifting the epiglottis, even though Magill had

clearly described the technique of paraglossal

straight-blade laryngoscopy (Scott 2009). Macin-

tosh disagreed with the midline approach. In his

1943 textbook, he wrote that using a straight

blade in the midline could “make the tongue

bulge over the blade and obscure the view”(Mac-

intosh 1943). Poor straight-blade technique hin-

dered laryngoscopy and set the stage for

Macintosh to develop his famous laryngoscope

(Scott 2009).

Macintosh experimented with different

devices and found one day during tonsillectomy

surgery, “Opening the mouth with the Boyle-

Davis gag, I found the cords perfectly displayed

...before the morning had ﬁnished”(Scott 2009),

The “Boyle-Davis gag”was a curved metal

device placed in the mouth and used to visualise

the oropharynx and stabilise the tonsils for

surgery. He got his assistant to solder this

“Boyle-Davis gag”onto a laryngoscope

blade, and thus the Macintosh blade was born.

Macintosh discovered that the key to achiev-

ing this “perfect display”of the laryngeal inlet

was his new method of indirectly elevating the

epiglottis: “the important point being that (with a

blade shorter than other laryngoscopes) the tip

ﬁnishes up proximal to the epiglottis”(Jephcott

1984). Macintosh was not the ﬁrst to observe that

the blade did not need to go beyond the epiglottis,

he was however the ﬁrst to suggest the standard of

placing the blade tip within the vallecula (Scott

2009). He illustrated the fact that when the laryn-

goscope tip is placed in the vallecula and is “lifted

... the epiglottis, because of its attachment to the

base of the tongue, is drawn upwards and the

(entire) larynx comes into view”(Macintosh and

Bannister 1943) This new technique of indirectly

elevating the epiglottis still remains the preferred

method of direct laryngoscopy worldwide (Scott

2009).

Despite acknowledging the obvious curve of

his own laryngoscope, Macintosh believed that

people focused too much on the curve of the

blade. He stated that “The precise shape or

curve of the blade does not seem to matter much

provided the tip does not go beyond the epiglot-

tis”and providing the technique of the user was

adequate (Macintosh 1944). Macintosh eventu-

ally settled on a curve that mimicked that of the,

then-popular, Magill’s endotracheal tube. In fact,

in the early days Macintosh laryngoscopes could

be bought with various curves, or even straight,

illustrating that the point was not the curve, but

rather the method of indirectly lifting the epiglot-

tis with a shorter blade (Scott 2009).

The Macintosh and the Miller laryngoscope

formed the cornerstone of anaesthetic airway

management until modern times, and indeed are

still the predominant laryngoscopes used in much

of the world today (Fig. 4.3). There are a small,

but signiﬁcant number of people whom obtaining

an adequate view of the glottis is either extremely

difﬁcult or impossible with the Macintosh and

Miller laryngoscopes. As a result, there have

been several attempts to modify these in order to

overcome these situations (Hagberg 2018).

68 D. King and A. Blair

Fig. 4.3 Modern Miller (top) and Macintosh (bottom)

laryngoscope blades

Before considering the modiﬁcations, it is use-

ful to understand the components of the modern

laryngoscope.

4.2.4 Modern Laryngoscope

The basic structure of a laryngoscope consists of a

blade, a handle and a light source (Hagberg

2018). The main shaft of the blade is known as

the spatula. Its function is to displace and com-

press the tongue and soft tissues. This allows the

oral and pharyngeal axis to be aligned, providing

a direct line of site between the laryngeal inlet and

the operators eye (Figs. 4.4,4.5, and 4.6). The

blade can be straight or curved as previously

discussed. The blade also comes in different

sizes, from size 0 (used in neonates) to size

4 (used in large adults) (Fig. 4.7).

The upward projection on the top of the blade

is known as the ﬂange (Figs. 4.4,4.5, and 4.6). On

the Macintosh laryngoscope the ﬂange is a

reverse-Z shape, and functions to help displace

and hold the tongue out of the ﬁeld of vision. The

tip of the laryngoscope (sometimes called the

beak) is blunt and thickened in order to prevent

any trauma to the soft tissues. The light source is

located in close proximity to the tip, and is

provided either directly by a lightbulb, or indi-

rectly via ﬁbreoptics from a light source at a

distance. The handle of the laryngoscope contains

the batteries for the light source and comes in

various sizes. These are standard, paediatric

(which is thinner to improve the balance with

small paediatric blade) and a short handle

(to improve ease of intubating obese patients or

those with large breasts). The blade attaches onto

the handle via a hook at the base of the blade. This

base also contains an electrical contact that

provides power from the battery to the light

(Figs. 4.4,4.5,and4.6).

Fig. 4.4 Modern Macintosh blade and handle lateral view

Despite the enduring popularity and success,

even until present day, of the Macintosh and

Miller largyngoscopes, there was a small but sig-

niﬁcant proportion of the population upon whom

Fig. 4.5 Modern Macintosh blade and handle

obtaining an adequate view of the vocal cords

was either extremely difﬁcult or impossible

using these devices. There has over the years

been numerous attempts, through functional

adaptions to the “basic”Macintosh or Miller

laryngoscope to try and rectify this.

4 The Evolution of Equipment and Technology for Visualising the Larynx and Airway 69

Fig. 4.6 Modern Macintosh blade (posterior view). Note

reverse z-shaped ﬂange

Fig. 4.7 Modern Macintosh blade size (from top to bot-

tom) 4, 3, 1, and 0

4.2.5 Macintosh Related Curved

Blades

One of the ﬁrst adaptions to Macintosh’s blade

was by Dr. Parrott in 1951. He lengthened the

blade by around 2.5 cm and created a more grad-

ual curve. This was in an attempt to facilitate

intubating patients with prominent teeth or a

receding jaw, while the additional length was

useful in larger patients (Parrott 1951). The

Parrott laryngoscope was otherwise identical to

the Macintosh.

While it was recognised that the straight and

curved blades each had their own speciﬁc

advantages, Drs. Bowen and Jackson created a

laryngoscopy blade in 1958 that they hoped could

be successful in all difﬁcult scenarios. The blade

was almost straight with a signiﬁcant curve to the

distal aspect, and the tip was split. This split

allowed it to be placed deep into the vallecula,

and in theory more effectively lift the epiglottis

(Bowen and Jackson 1952).

A mirror-imaged version of the Macintosh

laryngoscope exists, fairly oddly named the

“left-handed laryngoscope”, as it is conversely

held in the user’s right hand. It is identical in

blade shape as the regular Macintosh laryngo-

scope, however the ﬂange is orientated in the

opposite direction. This could be used in patients

with oropharyngeal abnormalities necessitating

placement of the endotracheal tube in the left

side of the patients’mouth (Pope 1960).

The distal spatula of a few blades has been

reported as having a sharper curve than the Mac-

intosh. Most noteworthy was one produced by

Gubuya-Orkin, which was ﬁrst described in

1959. This was distinctive for having an

S-shaped blade and a 3 cm bendable distal portion

which they described bending it through a range

of 15–45°. The ideal intubating angle, according

to the authors, is 35°from horizontal, which

enables the instrument to lift the epiglottis indi-

rectly (Gabuya and Orkin 1959). The blade,

which was discovered to be particularly success-

ful in some cases when laryngoscopy using the

Macintosh blade had failed, may have been the

prototype for other blades having a distinct set or

adjustable distal curvature (Hagberg 2018).

70 D. King and A. Blair

A signiﬁcant improvement was made to the

original Macintosh laryngoscope by Raez

(1984). He altered the mid-blade by ﬂattening

the curve slightly and made slightly concave.

These combined alterations should lessen the

“crest-of-hill”effect, in which the convexity at

the midblade can obstruct the direct line of vision

between the user and the laryngeal inlet. This

blade is known as the Improved view Macintosh

(Raez 1984).

4.2.6 Miller Related Straight Blades

There have been numerous attempts at alterations

to the classic Miller straight blade. Most notably

are the Soper, the Wisconsin and the Phillips

blades. The Soper was introduced in 1947,

which was largely straight. It was produced in

response to difﬁculty in lifting a long “ﬂabby”

epiglottis by the Macintosh blade (Soper 1947). It

had a small transverse slot cut into the distal blade

in order to prevent the epiglottis from slipping off

the blade. The Wisconsin is entirely straight with

a C-shaped ﬂange that gradually increases in size

from proximal to distal (Hagberg 2018). The

Phillips blade was released in 1973 and is a com-

bination of the slightly curved tip of the Miller

and the shaft of the Jackson blade (Phillips and

Duerksen 1973). The Soper, the Winsonsin and

Phillips blades are all still in commercial use

today.

4.2.7 Levering Tip Laryngoscope

The levered tip laryngoscope (Corazzel-

li-London-McCoy or CLM blade) was initially

produced as an adaption to the Macintosh curved

blade in 1993, although it is now readily available

in both curved and straight blade conﬁgurations.

These blades have a distal hinging tip that can be

activated by a spring-loaded lever attached to the

laryngoscopes handle. Pressing the lever elevates

the tip, which is the ﬁnal 2.5 cm of the blade, by

around 70°(Figs. 4.8 and 4.9). This tip can act in

the vallecula, providing good contact with the

hyoepiglottic ligament, allowing lifting of the

epiglottis. This can be particularly useful in

situations of limited neck movement, where

force through the blade could be detrimental, or

situations of limited mouth opening (McCoy and

Mirakhur 1993). In theory less force is required

when using a CLM blade, and therefore less sym-

pathetic stimulation is seen (increasing heart rate

or mean arterial pressure) (McCoy and Mirakhur

1995). Some recent studies have shown using the

CLM blade and engaging the tip may in fact

worsen an otherwise easy view (Tuckey et al.

1996). This is thought to be due to the fact that

in these “easy views”the tip would easily and

fully engage the vallecula and lift the

hyoepiglottic ligament. Further activation of the

handle and tip in fact causes posterior displace-

ment of the blade, as there is no further anterior

displacement of the hyoepiglottic ligament possi-

ble, which causes worsening of the view (Levitan

and Ochroch 1999). The CLM blade has been the

most recent signiﬁcant adaption to the classical

indirect laryngoscopes.

Fig. 4.8 CLM laryngoscope

4 The Evolution of Equipment and Technology for Visualising the Larynx and Airway 71

Fig. 4.9 CLM laryngoscope with hinging tip activated

4.2.8 Return to Indirect

Laryngoscopy

While the Macintosh and Miller blades ensured

that direct laryngoscopy was by far and away the

most common method of endotracheal intubation,

there have been numerous rigid indirect

laryngoscopes introduced over the last 60 years.

These rigid indirect laryngoscopes allow a view

of the laryngeal inlet and vocal cords indirectly

through the use of mirrors, ﬁbreoptic cables, or

video cameras (Hagberg 2018). They provide an

advantage as they can “look round the corner”,

therefore potentially improving an otherwise dif-

ﬁcult intubation. The structure of these indirect

laryngoscopes can vary from a slightly adapted

Macintosh blade to an entirely distinctive design

altogether.

While the impression of these indirect

laryngoscopes is that they are modern inventions,

in fact the ﬁrst widely available device was pro-

duced by Siker in 1959. He produced a blade with

an incorporated mirror, which can be used to

identify the epiglottis and laryngeal inlet if they

cannot be directly visualised. Copper wiring

attaches the mirror to the blade, allowing heat

conduction in an attempt to prevent the mirror

from steaming up. The mirror inverts the image,

meaning that the process of tracheal intubation

takes a large amount of speciﬁc practice with the

Siker device (Siker 1956).

McMorrow and Mirakhur developed a similar

mirrored device. It was an adaption of the regular

CLM laryngoscope but was unique in that

activating the lever not only elevated the tip, but

deployed a mirror posteriorly and inferiorly to the

blade (McMorrow and Mirakhur 2003).

4.2.9 Prisms in Laryngoscopes

The use of prisms in laryngoscopes was not a new

concept, as Kristen had done in the late nineteenth

century, however Huffman (1968) re-introduced

the concept. He attached a triangular prism to a

Macintosh blade, allowing refraction of the light

through 30 degrees, and allowing the user to “see

round the corner”(Huffman 1968). This allowed

improvement in obtainable view in particularly

challenging intubations, and also allowed less

force to be used when trying to obtain a view.

He subsequently attached a second prism more

proximally, creating a second device that allowed

refraction of light through 80°.

The Airtraq device was the most commercially

and clinically available Prism-assisted device.

The blade of the Airtraq is designed to be

“anatomically shaped”, and thus at a more acute

angle than the Macintosh blade. The concept was

aimed at creating a device that would provide a

view of the laryngeal inlet while not requiring

alignment of the oral, pharyngeal, and tracheal

axes (Hagberg 2018). This allows easy

visualisation of the glottis while intubating. As

the Airtraq is able to “look around the corner”of

the tongue to obtain a laryngeal view while using

minimal “lifting force”, it is suggested it provides

a clinical advantage in certain scenarios. These

include patients with an anterior larynx (a term

used within to describe the phenomenon of the

laryngeal inlet appearing more anterior to the line

of sight during laryngoscopy) or restricted neck

movements from any cause (fractures and

immobilisation collars, radiotherapy, burns etc.).

The Airtraq consists of two adjacent channels.

The “viewing channel”consists of various lenses

and prisms and terminates at the proximal eye

piece on the laryngoscope handle. The adjacent

channel acts as a “housing channel”for the endo-

tracheal tube and allows easy advancement of the

tube down the channel and through the vocal

cords, under vision via the eyepiece. With the

tube in position, it can be separated from the

Airtraq device by “peeling”. The battery-powered

light source is found at the tip of the blade, as is a

small heater, which heats and prevents fogging of

the distal lens (Hagberg 2018).

72 D. King and A. Blair

4.2.10 Fibreoptics

The largest and most signiﬁcant development

with regard to laryngoscopy and intubation over

the last 70 years has without doubt been the

introduction of ﬁbreoptics. Fibreoptic scopes

were initially developed in the 1950s for use as

endoscopes. Over time, bronchoscopes became

more readily used, but it was not until the 1980s

when ﬁbre-optics were utilised in anaesthetic

practice as part of rigid indirect laryngoscopes

(Hagberg 2018).

Fibre-optic cables transmit their light by

means of total internal reﬂection. This is the pro-

cess by which light in a medium is completely

reﬂected back into the medium by its surround-

ings. All light is refracted (or “bent”) to a degree

when it passes between two differing substances.

This is because the substances have a different

refractive index. A commonly seen example of

this is viewing an object that is half submerged in

water. Water and air have different refractive

indices so the object will appear bent at the

point it enters the water. Total internal reﬂection

occurs when refraction is so great that the whole

beam is bent (or reﬂected) back into the original

material. This occurs when the angle between the

light source and the boundary is greater than the

critical angle.

Aﬁbre-optic cable transmits information as

light (photons) via incredibly thin (around

20 μm in diameter) strands of glass or plastic

known as optical ﬁbres. There are around

15,000 of these glass ﬁbres in a ﬁbre-optic

scope. Fibreoptic devices have bundles of around

15,000 glass ﬁbres, each about 20 μm in diameter.

These strands make up the core of the ﬁbre-optic

cable. The core is surrounded and encased by a

layer of a different type of glass known as the

cladding. It is the boundary between the core and

the cladding that allows for total internal reﬂec-

tion. The cladding is surrounded by a protective

coating, as well as strengthening and supportive

Kevlar ﬁbres in modern devices.

These ﬁbre-optic scopes allow rapid and

clear transmission of pictures, which has

revolutionised medical practice, from endoscopes

to view the gastrointestinal tract to bronchoscopes

which allow ﬁbre-optic intubations.

Rigid indirect ﬁbre-optic laryngoscopes were

the biggest evolutionary change in anaesthetics

since Mactinosh’s creation. While the popularity

of the laryngoscopes individually was never

great, they signalled a signiﬁcant advancement

in the technology in anaesthetic practice. They

were introduced in the late 1980s in the form of

the Bullard Laryngoscope. It features a rigid

L-shaped blade and incorporates ﬁbre-optic

bundles that transmit the image to the proximal

eye piece (Kastnelson and Straker 1996). The

blade of the Bullard laryngoscope is anatomically

shaped, allowing for indirect visualisation of the

laryngeal inlet, without alignment of the oral,

pharyngeal, and laryngeal oriﬁces. It was

conceived as a laryngoscope that is useful in

patients who are difﬁcult to intubate, particularly

with minimal neck movement. It can also be used

for awake intubation in patients as minimal

“lifting force”is required for intubation. The

Bullard laryngoscope features a “work channel”

which lies posterior to the blade and can be used

for suction as well as local anaesthetic or oxygen

administration during laryngoscopy. The Bullard

laryngoscope also features an attachable metal

stylet, which can be used with a pre-loaded endo-

tracheal tube to facilitate intubation. The eyepiece

can either be used directly by the user or can be

attached to a video camera for display on a large

screen (Borland and Casselbrant 1990).

The UpsherScope is similar in design to the

Bullard Laryngoscope, however, consists of a

J-shaped rather than L-shaped blade (the blade

has less of an acute angle to it), which is also

thinner and ﬂatter. Instead of an attached stylet,

the UpsherScope has a C-shaped channel poste-

rior to the blade which is designed to help guide

the endotracheal tube through the cords when

intubating. It is simpler in design when compared

to the Bullard laryngoscope, as it has no acces-

sory channel for suctioning or administration of

drugs. It is otherwise similar to a handle and ﬁbre-

optic viewing ﬁbres that connect to an eye piece

(Pearce and Shaw 1996).

4 The Evolution of Equipment and Technology for Visualising the Larynx and Airway 73

The WuScope is the third of the common rigid

ﬁbreoptic scopes that were seen in clinical prac-

tice. It is similar in concept to the UpsherScope

and the Bullard Laryngoscope, however differs in

its design. It consists of two components: a

removable ﬂexible ﬁbreoptic scope and a rigid

blade laryngoscope structure. The blade itself

consists of a handle, and a hollow tubular blade

that contains two internal channels. One of these

channels is the guiding passage for the endotra-

cheal tube, while the other houses the ﬁbreoptic

endoscope. The tubular structure was designed in

order to protect the endoscopic ﬁbres from

secretions or blood, as well as hold soft tissue

structures of the airway out of the way. The

WuScope also incorporates an oxygen channel

next to the endoscopic ﬁbres. The blade attaches

to the handle of the device at 110°which was to

allow for easy entry into the oropharynx (Wu and

Chou 1994).

4.2.11 Flexible Fibre-Optic Scope

Intubation

While rigid ﬁbre-optic laryngoscopes are still a

relatively recent invention, ﬁbre-optic intubating

bronchoscopes have been around since 1967

when Dr. Peter Murphy ﬁrst demonstrated the

procedure using a surgical choledochoscope.

Quickly the practice of ﬁbreoptic intubating

bronchoscopes became more widespread, initially

for normal intubations, but soon for difﬁcult

airways. Due to its obvious advantages compared

to rigid devices, the ﬂexible intubating broncho-

scope became the gold standard throughout

anaesthetic practice for intubating patients with

difﬁcult airways and cervical spine high-risk

patients. It also became an extremely useful

diagnostic tool for assessing potentially difﬁcult

airways, conﬁrming endotracheal tube placement

(in particular double lumen tubes), as well as

therapeutic management of foreign bodies/

secretions (Hagberg 2018).

The obvious main advantage of the ﬂexible

intubating bronchoscope is the minimally inva-

sive nature, so the intubation can therefore be

done awake with no muscle relaxants.

All ﬂexible ﬁbre-optic bronchoscopes have the

same basic components: the body, the insertion

cord and the ﬂexible tip.

The body consists of the handle of the device

on which is attached several key features. It

contains an eyepiece and lens, or on more modern

devices, a video output adapter or actual video

screen. The handle also contains a light source

which is either battery operated for portability or a

connection cable for an external light source. The

handle also contains a single plane control lever

for controlling the direction of the tip (either up or

down), as well as an attachment point for a suc-

tion channel (Hagberg 2018).

The body handle is attached to the insertion

cord which is the part that enters into the patient.

It is a thin ﬂexible cord that passes into the

patients’airway and be inserted orally or nasally.

It contains several components that allow

visualisation of the structures of the airway. The

ﬁbreoptic light bundle transmits light down to the

tip from the light source, while the ﬁbreoptic

image bundle transmits the image in the form of

light from the tip to the eye piece. The light

bundles are made up of anywhere between

10,000 and 50,000 glass ﬁbres, each 8 or 9 μm

in diameter. Each of these strands is surrounded

by a layer of cladding, as discussed earlier. Also

contained withing the insertion tube are angula-

tion wires. These run the length of the insertion

tube and connect the control lever to the tip of the

ﬁbreoptic device. On ﬂexing the control lever,

these move the tip through a vertical plane

allowing the user to direct the ﬁbre scope. The

ﬁnal component of the insertion tube is a suction

channel that runs from the tip to the suction port

on the handle. This channel can alternatively be

used to administer medications (such as local

anaesthetics) or used as a biopsy port.

74 D. King and A. Blair

The ﬁnal aspect of a ﬁbre-optic scope is the

ﬂexible tip which contains a lens for focusing the

light entering the scope.

Fibre-optic intubations can either be done

orally or nasally. Oral ﬁbreoptic intubation is

generally seen to be a more challenging proce-

dure as the tongue tends to collapse to the back of

the oropharynx, even in an awake patient, making

navigation with the ﬁbreoptic scope more difﬁ-

cult. Several airway accessory devices have been

introduced to assist with oral ﬁbreoptic

intubations. These devices, which include the

Berman Intubating Airway (Figs. 4.10 and 4.11)

and the Williams Airway Intubator, sit in the

oropharynx and help guide the ﬁbreoptic scope

down towards the larynx and vocal cords. They

also function to hold the tongue and soft tissues

out the way to allow passage of the scope and

have a “breakaway slit”to allow them to be

peeled off the scope once it has passed through

the vocal cords. Once this accessory device has

been removed, the endotracheal tube, which had

been pre-loaded onto the ﬁbreoptic scope before

starting, is then railroaded down the scope and

through the cords.

Fig. 4.10 McGrath videolaryngoscope right lateral view

Fig. 4.11 McGrath videolaryngoscope, left lateral view

Nasal ﬁbreoptic intubation is often seen as

simpler as the nasal passage helps guide the

scope towards the vocal cords. The size of endo-

tracheal tube that is used is restricted, however,

generally to a size 6, due to the relatively small

size of the nostrils. This has implications for

ventilation, and particularly with ongoing man-

agement in intensive care afterwards, if applica-

ble. For this reason, the user may often the elect to

afterwards do an airway exchange for an oral

endotracheal tube once nasal ﬁbreoptic intubation

has been achieved.

Fibreoptic intubations can be done either

awake or unconscious. They major use beneﬁt

of ﬁbreoptic intubations is in those patients with

known or suspected difﬁcult airways, most

practitioners will elect to use awake ﬁbreoptic

intubations to keep the patient spontaneously

breathing throughout should any difﬁculties arise.

Due to this ability to “bail out”from the intu-

bation, awake ﬁbreoptic intubation (AFOI) is the

most risk-free method of intubation, so there does

not in fact need to be a speciﬁc indication to use

it. There are speciﬁc situations where AFOI is

speciﬁcally indicated.

4 The Evolution of Equipment and Technology for Visualising the Larynx and Airway 75

•It remains the gold standard for any patient

with a known or expected difﬁcult airway as

mentioned, speciﬁcally for patients with oro-

pharyngeal malignancies. It is not particularly

effective for patients with tumours or strictures

of the glottic or subglottic regions, however.

•Any patients with cervical spine instability.

•Those at risk of intubation trauma, e.g. loose

teeth.

•Avoidance of aspiration in very high-risk

patients.

•Allergy to muscle relaxant.

•Placement of double lumen tubes and

tracheostomies.

•For cardiovascular stability—as induction

once endotracheal tube is in situ is generally

inhalational.

One of the biggest restrictions to the use of

ﬁbreoptic scopes is cost. Re-usable ﬁbreoptic

scopes produced by Olympus with accompanying

screen and equipment costs upwards of £20,000

which is prohibitively expensive for many

departments (Hagberg 2018). A cheaper alterna-

tive is the Ambu aScope, which is a disposable

ﬂexible scope utilising a camera and LED light

source. The images are electronically transmitted

to a screen rather than through the use of

ﬁbreoptics. However, it is a single use device, so

waste is increased and the image quality is infe-

rior to a ﬁbreoptic scope.

There are few absolute contraindications to

AFOI, however a uncooperative patient will

make attempts at that method pointless. As it is

a more specialised method of intubating,

untrained staff also precludes AFOI. Near total

airway obstruction (unless used for diagnostic

purposes) is also a contra-indication, as would

an allergy to local anaesthetics (Hagberg 2018).

4.2.12 Videolaryngoscopes

While rigid ﬁbreoptic laryngoscopes were

initially considered a niche development, they

introduced the world of anaesthetics to the

concept of videolaryngoscopes. These devices,

however, remained fairly peripheral following

their invention, as ﬂexible ﬁbreoptic

bronchoscopes were by far the most popular

devices for difﬁcult airways. The trusted Macin-

tosh laryngoscope was used for patients with

“regular”airways.

The popularity and availability of the videolar-

yngoscope increased signiﬁcantly from the early

2000s due to yet more technological

advancements. The signiﬁcant draw backs of the

rigid indirect ﬁbreoptic laryngoscopes discussed

were the signiﬁcant cost of the ﬁbre-optics, as

well as their bulky size. A Macintosh laryngo-

scope was easily transportable in the users’

pocket, whereas the ﬁbreoptic scopes were less

portable, especially if being used with a screen

(Chemsian et al. 2014).

The invention and availability of LCD (liquid-

crystal display) screens, LED (light-emitting

diode) lights, and CMOS (complementary

metal-oxide semiconductor) video chip technol-

ogy which are utilised in modern videolaryn-

goscopes has made these devices much cheaper,

portable, and easier to use (Hagberg 2018).

Videolaryngoscopes tend to be categorised

according to the shape of their blade. These

categories are “Macintosh style blades”, and

“acute angulated blades”. There often is a third

category of videolaryngoscopes added which is

“channelled devices”(Chemsian et al. 2014).

4.2.13 Macintosh Style Blades

These devices integrate video technology onto a

modern Macintosh-styled laryngoscope. They

have the added advantages of familiarity of

blade shape, as well as being able to use the

device for direct laryngoscopy if there was a

failure with the video technology.

The Mcgrath laryngoscope is a portable

videolaryngoscope, which is battery powered

and has a small adjustable screen attached to the

handle. It comes with the standard Macintosh

sizes of disposable blades from 0 (baby) to size

4 (large adult). It also comes with an X-blade,

which is an acute angulation blade for difﬁcult

airways, making the Mcgrath an all-round

videolaryngoscope (Shippey et al. 2008).

76 D. King and A. Blair

Due to its signiﬁcant advantages of relatively

low cost and portability, it has become the most

widely utilised videolaryngoscope (Niforopoulou

et al. 2010).

The C-MAC, which was produced by

Storz medical, was the ﬁrst videolaryngoscope

to be widely commercially available. It also

consists of a Macintosh laryngoscope with an

incorporated video technology. It differs from

the Mcgrath laryngoscope, in that the handle of

the laryngoscope is attached to a distant larger

screen via a cable. This provides the advantage

of everyone in the room being able to see the view

which helps in situations of teaching as well as

patients with a difﬁcult airway. The blade of the

C-MAC is interchangeable from an infant to adult

size 4 (Chemsian et al. 2014).

The GlideScope direct was the original

GlideScope design with a metal,

non-interchangeable size 3.5 Macintosh blade.

The handle of the laryngoscope is attached to a

distant monitor, similar to the C-MAC, and the

device can be used for direct laryngoscopy, or

indirect using the video function.

4.2.14 Acute Angulated Blades

Devices that incorporate a more acute angulation

to their blades compared to the standard Macin-

tosh blade. This acute angulation has been seen in

several studies to improve glottic visualisation

with minimal patient c-spine ﬂexion. This has

been seen to be useful in patients with known

difﬁcult airways, and those with an anterior posi-

tioned larynx. The drawback with these acute

angled blades is that while the view is often

improved, passing the endotracheal tube through

the cords is more challenging due to the acute

angulation, and an intubating stylet is usually

required. Another major drawback is they cannot

be used for direct laryngoscopy should the video

equipment fail.

The GlideScope with the acute

hyperangulation blade of 60°, and otherwise has

similar equipment to the non-angulated

GlideScope. It comes with a speciﬁcally designed

rigid stylet (GlideRite stylet) for use during

intubating due to the acute angulation of the

blade.

Mcgrath Series 5 is a portable laryngoscope

with the small screen attached to the handle simi-

lar to the standard Mcgrath, however with an

acute angled blade. The blade portion is fully

detachable and its connection position on the

handle can be altered. This clever feature allows

for the blade to be ﬁrst inserted into the patients’

mouth, then connected to the handle, which is

potentially useful in those obese patients or

those with large breasts (Niforopoulou et al.

2010).

C-MAC D-Blade is an acutely curved version

of the original, produced by Storz medical in

2010. The blade is curved, and functions, in a

similar fashion to the GlideScope, and requires

the use of an intubating stylet (Cavus et al. 2011).

King Vision laryngoscope was only released

in 2017, so is one of the newest devices available.

It has a similar design style to the Mcgrath in that

it has a small screen attached at the top of the

handle and is fully portable. It has two types of

disposable blades, the standard aBlade which is

an acute angle blade, or the channelled blade

(Kriege 2017).

4.2.15 Channelled Devices

These devices use a side channel to direct the

pre-loaded endotracheal tube through the vocal

cords. With the endotracheal tube pre-loaded,

there is no requirement for stylets or gum elastic

bougies to assist with intubation.

The King Vision device as mentioned has an

option of a channelled blade, and so can be

categorised as a channelled device.

The Airtraq device, as discussed earlier in the

chapter, while not strictly a video-laryngoscope,

is often incorrectly categorised as a channelled

videolaryngoscope. While not containing any

video technology, the camera can be attached

to the eyepiece of the device for display on a

screen.

4 The Evolution of Equipment and Technology for Visualising the Larynx and Airway 77

The Pentax AWS is a true videolaryngoscope

with a side channel for guiding of the endotra-

cheal tube. It is portable, with the screen attached

to the handle. The screen displays a target mark

which is aligned with the laryngeal structures, and

the endotracheal tube is then advanced through

the cords. As with all the channelled devices,

when the endotracheal tube is through the vocal

cords, it needs to be “peeled”off the laryngo-

scope in a lateral motion to allow removal of the

device.

4.3 Conclusion

There have been clear and marked improvements

in the available technology used for tracheal

intubations, particularly over the last 40 years.

These improvements are reﬂected in signiﬁcant

improvement in adverse outcomes and generally

in patient safety with regard to intubations (Li and

Warner 2009). Awake ﬁbre-optic intubation

quickly became the gold standard for patients

with known or expected difﬁcult airways. There

were pitfalls however, mainly with regard to cost

and requirement of an experienced team in AFOI.

The advent of videolaryngoscopy over the last

20 years has changed the landscape dramatically.

It is now well established that videolaryngoscopy

improves the achievable laryngeal view when

compared to direct laryngoscopy. Numerous

recent studies have shown extremely high rates

of success using Mcgrath (95% success rate)

(Noppens and Möbus 2010), Pentax (95% suc-

cess rate) (Asai et al. 2010), and GlideScope

(96% success rate) (Aziz et al. 2011) in either

conﬁrmed or suspected difﬁcult airways. The

GlideScope even showed a 94% success rate as

a rescue device following a failed attempt at direct

laryngoscopy. This has been incorporated into

studies, which suggest videolaryngoscopes

showed no beneﬁt when compared to direct lar-

yngoscopy on patients with regular “easy”

airways.

So, with videolaryngoscopy being as effective

in “routine”airways, and clearly superior in difﬁ-

cult airways the expectation may be that

videolaryngoscopy continues to proliferate in

popularity and will in time fully displace direct

laryngoscopy as the primary method of endotra-

cheal intubation. Given that videolaryngoscopy

has a clear beneﬁt over direct laryngoscopy in

teaching, due to the potential for a shared view

between teacher and trainer, as well as its use in

awake patients (using local anaesthetics and acute

angulation blades), then that time may be not too

far in the future.

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Technology has allowed the capture of the

The Impact of Technological Innovation

on Dentistry 5

Richard Zimmermann and Stefanie Seitz

Abstract

Technology has revolutionized the way

dentists are able to treat their patients. These

technological advances have paved the way

for the creation of virtual patient models

utilizing these 3-dimensional intra-oral patient

models, cone bean computer tomography

(CBCT) radiograph scans, extraoral

3-dimensional scans, and jaw motion tracings

to create a patient-speciﬁc model. These

models are advantageous in planning surgical

treatments by providing 3-dimensional views

of vital anatomical structures to accurately

identify the location, size, and shape of a struc-

ture or defect in order to plan accordingly.

Virtual augmentation of either hard tissue

(bone) and/or soft tissue (i.e., gingiva) can

also be accomplished.

dynamic motions of the jaw and combined

them with the virtual patient to develop perma-

nent restorations in harmony with the patient’s

orofacial complex. With the introduction of

new technology in the realm of digital den-

tistry, patient care is being brought to a new

and higher level. This creates a level of more

optimal care that a dentist can deliver to

patients.

R. Zimmermann · S. Seitz (✉)

Department of Comprehensive Dentistry, UT Health San

Antonio, San Antonio, TX, USA

e-mail: zimmermann@uthscsa.edu;seitz@uthscsa.edu

#The Author(s), under exclusive license to Springer Nature Switzerland AG 2023

E. Abdel Meguid et al. (eds.), Biomedical Visualisation, Advances in Experimental Medicine and

Biology 1406, https://doi.org/10.1007/978-3-031-26462-7_5

Keywords

Digital dentistry · 3-dimensional model ·

Virtual model · Virtual patient · Patient-

speciﬁc model · Jaw movement tracing

5.1 Introduction

Advances in the area of digital dentistry have

propelled the profession of dentistry into a

completely new era. Historically, impressions of

patients’teeth were taken using an alginate mate-

rial and poured in dental stone to create models.

These models were then utilized, along with

2-dimensional radiographic images, to evaluate

bony structures. However, dentists now use

wands that intraorally scan the patient and are

able to create 3-dimensional virtual models of

the patient’s teeth. These 3D models from the

scan allow for the beginning of the creation of

virtual patients—both general models for educa-

tional use and patient-speciﬁc models (Joda et al.

2019). The creation of these comprehensive

patient models can be accomplished with the

combination of any number of components,

including 3-dimensional intra-oral patient models

or surface scans, cone bean computer tomography

(CBCT) radiograph scans, 3-dimensional

extraoral scans, and jaw motion tracings.

Deﬁnitive advantages are associated with

these patient-speciﬁc models when planning vari-

ous surgical treatments because they are able to

provide the dentist with 3-dimensional views

of vital anatomical structures that may have not

been able to be visualized on a traditional

2-dimensional radiograph. An enormous beneﬁt

associated with these new 3-dimensional views is

that the models are to scale, allowing the surgeon

to plan treatment effectively by providing

enhanced accuracy in identifying the location,

size, and shape of a structure or defect. Whether

planning surgery on the maxilla (upper jaw) or

mandible (lower jaw), the surgeon must be cogni-

zant of the vital structures, such as blood vessels

and nerves, that are located in these areas. If these

structures are injured, it could result in extreme

and/or prolonged harm to the patient. The knowl-

edge of the location of these defects enables the

surgeon to alter the plan to be able to lessen the

effects the defect can have on the surgery. For

example, one can virtually examine the complex

anatomy of a sinus, location of various septa, and

various neurovascular bundles of a patient prior

to performing the actual surgical procedure. This

translates into decreased surgical time as the sur-

geon is more familiar with the patient’s anatomy

prior to beginning the procedure. The need for

virtual augmentation of either hard and/or soft

tissue during a surgery can also be determined

based on these virtual models, along with the

ability of the surgeon to attain an accurate mea-

surement of the amount of grafting material that

will be utilized for the surgery.

80 R. Zimmermann and S. Seitz

In the area of restorative dentistry, the ability

to conduct a 3-dimensional oral reconstruction of

the patient’s dentition is incredibly useful. This is

accomplished by combination of intra-oral scans,

facial photographs, and 3D radiographic images

to create a “virtual model”of a patient. These

virtual models allow the development of perma-

nent restorations that are in harmony with the

patient’s orofacial complex—both esthetically

and functionally. Speciﬁcally in esthetic den-

tistry, the virtual patient enables the dentist to

evaluate the shape and position of the patient’s

teeth relative to their facial structure and create a

virtual simulation of proposed restorations

(Garcia et al. 2018). This virtual mock-up is

incredibly useful for communication between

the patient, dentist, and laboratory technician.

Historically, the fabrication of dental restorations

was created on an analog articulator—a mechani-

cal device on which the dentist would mount

upper and lower jaw models to simulate the

patient’s functional jaw movement. However,

this device is limited by linear movements, so it

does not completely model the patient’s function

because jaw movement occurs in multiple planes.

Nowadays, technology has allowed us to capture

the dynamic motion of the jaw and combine it

with the virtual patient to design patient-speciﬁc

restorations that are in synergy with the patient’s

actual jaw movements (Lepidi et al. 2020).

A promising area emerging in digital dentistry

is the utilization of diagnostic imaging of a

patient’s airway to aid in the diagnosis and treat-

ment of sleep apnea which affects approximately

1 billion people worldwide. Therefore, develop-

ment of these new technologies, both hardware

and software, in the realm of digital dentistry, is

bringing patient care to a new and higher level.

This tremendously facilitates the role of the den-

tist in providing more optimal care that a dentist

can deliver to their patients.

5.2 Creating the Virtual Patient

The creation of a virtual patient begins with a scan

of the patient’s teeth using the digital impression

system. The scan renders a 3-dimensional virtual

model of the teeth that can be created in two

different ways. The ﬁrst way involves scanning

the traditionally made stone models with either a

laboratory scanner or intra-oral scanner (IOS),

while the second way involves directly scanning

the patient’s oral cavity with the utilization of an

intra-oral scanner (Fig. 5.1).

Intra-oral scanning (IOS) has several distinct

advantages over the conventional impression

materials and techniques (Marques et al. 2021;

Rutkūnas et al. 2017). For patients, IOS provides

less discomfort by eliminating the need for

impression trays loaded with a considerable

amount of impression material that tends to elicit

the gag reﬂex which is overall unpleasant. The

second advantage which patients enjoy is the

decreased amount of time needed for the

3-dimensional model. An experienced clinician is

able to complete a scan in just a few minutes. For

the clinician, digital impressions allow for a

cleaner, faster, and more accurate representation

of the patient’s oral cavity. Utilizing the tradi-

tional method requires mixing and pouring dental

stone into the impression, along with a period of

time in which the dental stone must be set. After

which, trimming and cleaning the models are

needed. Also, legally, dentists are required to

keep patient models for a speciﬁc number of

years depending on where their practice is located

because they are considered a part of the patient

record. Virtual models facilitate this requirement

by allowing the models to be stored virtually and

not requiring an actual physical space. This is a

considerable beneﬁt to those clinicians with

thousands of patients in their practices.

5 The Impact of Technological Innovation on Dentistry 81

Fig. 5.1 Intra-oral scan of patient capturing both hard

(teeth) and soft (gingiva) tissues. Color is picked up by

the scanner

Intra-oral scanners are handheld devices that

typically consist of a wand with a camera in the

tip. The wands are moved to capture all angles of

the patient’s teeth, taking hundreds of pictures as

it progresses. The ability to scan is easily accom-

plished and dentists ﬁnd that there is a short

learning curve associated with it. While the tech-

nology used in these scanners varies, the result of

the pictures is a 3D mesh created from a point

cloud using a mathematical algorithm. This 3D

mesh is the virtual model of the patient’s dental

arch, capable of capturing both hard and soft

tissue (Fig. 5.2).

Intra-oral scanners are most often used to scan

a patient’s oral cavity while the individual is

sitting in the dental chair, which is the reason

they are often referred to as chairside scanners.

There are numerous scanners on the marker with

features that target different types of users. Some

of these scanners employ the technology that also

allows the capture of various shades and colors of

the anatomical structures and embeds that infor-

mation in the 3D mesh, creating life-like

recreations of the patient’s intra-oral cavity

(Haddadi et al. 2019). This colored mesh is an

advantage over the monochromatic lab scanners

because it allows for the visualization of a more

realistic virtual model of the patient, which can

aid in some diagnostic procedures.

Fig. 5.2 Intra-oral scan of patient illustrating the point

cloud that actually makes up virtual models

Chairside scanners embed 3-dimensional posi-

tioning coordinates into the digital scans during

the scanning process (Fig. 5.3). These coordinates

provide the relationship of the maxilla, to that of

the mandible, whenever the scans are viewed in

the software. However, this captured relationship

between the dental arches is static and only

represents when the two jaws are biting together

fully. While this is useful and might be all that is

necessary for some applications (for example—

smile analysis), other applications require the

dynamic movement of the mandibular arch with

respect to the maxillary arch (ex—fabrication of

nightguard for teeth grinding or a crown). Some

intra-oral scanners claim to capture this dynamic

movement; however, these scanners are only cap-

turing the beginning and end, which results in a

straight line of movement. This is not a true

representation of the human body and the move-

ment of the temporo-mandibular joint, which is

more ﬂuid and more curvilinear in nature

(Clayton et al. 1971).

82 R. Zimmermann and S. Seitz

Fig. 5.3 Implant planning software demonstrating the integration of virtual models (blue) to the CBCT to facilitate

planning

5.3 Utilization

of the 3-Dimensional Model

Cone-beam computed tomography (CBCT) is a

radiographic imaging technique that utilizes

round or cone-shaped x-rays to capture numerous

images as the scanner rotates around the patient.

This type of image creates certain advantages

over traditional 2D radiographs, including the

elimination of image magniﬁcation, overlapping

of anatomical structures, and image distortion.

These images are saved as Digital Imaging and

Communications in Medicine (DICOMs) ﬁles,

which consist of a collection of voxels—the unit

of graphic information that deﬁnes a point in three

dimensions. This radiographic imaging method

provides an accurate, 3-dimensional model of

hard tissue structures that is accomplished

through either “surface”or “volume”rendering.

Surface rendering utilizes gray scales of the

CBCT to create the model. The model can be

used for digitalization of 3D landmarks; it also

can be aligned with other 3D meshes, and even

enables the user to plan virtual osteotomies or

bone removal surgery. Volume rendering creates

the model by using the volume of voxels, to

which parameters are set based on the shading

algorithms. The disadvantage of this method is

the inherent inability to perform virtual surgeries

or the lack of the ability to align the model with

other 3D meshes. The utilization of CBCT scans

has become quite widespread and essential in

dentistry in all disciplines. Not only are they

used for diagnosis and treatment planning, but

also for the creation of detailed patient-speciﬁc

models and surgical guides (Baan et al. 2021).

For oral and maxillofacial surgery, the use of a

CBCT allows for the determination of the precise

location of anatomical features, such as

neurovascular bundles and tumors, as well as

impacted and supernumerary teeth. For Endodon-

tics, a specialty that focuses on teeth requiring

root canals and their associated pathologies,

CBCTs allow for the differentiation of apical

pathologies, visualization of fractured teeth, and

visualization of complex root morphology. For

periodontics, CBCTs can aid in the diagnosis of

furcation involvement due to bone loss, bone

defects around teeth, pathologies, and periodontal

cysts. The primary use of CBCTs in orthodontics

is to aid in assessing age, adolescent facial

growth, and disruptions in tooth eruptions.

CBCTs have created one of the largest impacts

in implant dentistry. The technology allows for

the measurement of the patient’s bone height and

width. It also enables localization of important

anatomical structures in the area of the planned

implant placement, as well as provides the capa-

bility to assess the need for additional surgical

procedures such as bone grafting.

5 The Impact of Technological Innovation on Dentistry 83

As in all radiographs, any metal present in the

oral cavity like amalgams (silver ﬁllings), crowns,

or existing implants can create radio-opacities.

These radio-opacities result in the decreased qual-

ity of the CBCT due to masking and distorting of

the anatomical structures and tooth morphology

being evaluated for diagnosis on the X-ray. Sev-

eral methods exist to decrease the amount of

scatter and improve the CBCT scan, however,

these do not help restore accurate morphology if

it has been distorted. To overcome discrepancies

within the oral cavity related to scatter, certain

software allows the merging, or fusion, of a

patient’s CBCT DICOMs with their

3-dimensional digital model created by the intra-

oral scan (Fig. 5.4).

This fusion creates an accurate 3-dimensional

model of the patient’s oral cavity, replicating both

hard and soft tissue in both radiographic and

dental model forms. In addition to the creation

of this fused virtual model of the oral cavity,

certain specialty areas of dentistry also require

the ability to relate the oral cavity with external

landmarks and features of the patient’s face. This

is of key importance when performing esthetic

reconstructions either through oral and

maxillofacial surgery or with dental restorations.

The simplest and most common approach at this

time is to align the intra-oral digital models to a

2-dimensional photograph of the patient. While

this accomplishes the goal of enabling the use of

facial features in treatment planning and restora-

tion design, there are inherent limitations.

Fig. 5.4 Patient stone models being mounted onto

articulator

The ﬁrst limitation, and the one with the larg-

est impact, relates to the alignment of a

3-dimensional dental model to a 2-dimensional

object, the patient photograph. Malalignment of

the patient models can result in a tilt of the occlu-

sal plane, the plane of the chewing surfaces of the

teeth, compared to the patient’s true occlusal

plane. This will have a negative inﬂuence on the

esthetic reconstruction which may not be possible

to recognize until treatment is rendered. For this

reason, this method is primarily used in esthetics

involving dental restorations; the proposed design

can be fabricated in temporary restorations,

allowing the dentist to identify the issue and cor-

rect it before proceeding to the fabrication of the

ﬁnal restorations. A second limitation is the abil-

ity to only view this type of virtual reconstruction

from the anterior, or front. One cannot rotate the

model to view from different angles due to the

constraints inherent with a 2-dimensional photo-

graph. The ability to rotate the images through

various viewpoints could be an advantage as it

would help evaluate the reconstructions in a more

realistic 3-dimensional environment.

84 R. Zimmermann and S. Seitz

In order to overcome these limitations

associated with 2-dimensional photographs,

3-dimensional scans of a patient’s head can be

obtained utilizing specialized equipment and can

be accomplished by two methods. The ﬁrst

method involves the integration of a facial scan-

ner within a CBCT machine. This method accu-

rately relates the 3-dimensional head scan to the

patient’s DICOMs, which can then be aligned to

intra-oral digital models. There is a limitation

associated with this method. It requires the patient

to obtain a full CBCT of the maxillofacial region,

which may be unnecessary if the patient is not

going to have any surgical procedures and results

in more radiation exposure than needed for the

patient. The second method involves the use of a

“handheld”scanner that captures a photorealistic

3-dimensional image of the patient’s head. For

alignment, additional scans are needed which

incorporate specialized markers that allow for

the accurate alignment to the 3-dimensional facial

scan. This method involves several more steps in

order to achieve optimum alignment but does not

expose the patient to unnecessary radiation.

While each of these methods has advantages and

disadvantages, both result in a 3-dimensional

model of the patient that can be viewed from

any angle, both with and without facial features.

This provides the dental provider an accurate and

more complete virtual patient model that allows

for comprehensive diagnosis and treatment

planning, something that had not previously

existed. It also leads to enhanced interdisciplinary

communication among various providers.

These virtual models discussed above are gen-

erally excellent tools for certain diagnosis and

treatment planning, interdisciplinary communica-

tion, and help keep a record of the patient as they

are presented in the dental chair. However, the

majority of dental treatment takes place in the

stomatognathic system, a functional environment

that is composed of skeletal structures, muscles of

mastication, the temporomandibular joint, and the

dental arches (maxillae/mandible). This system is

an incredibly dynamic environment, meaning it is

in motion for most of its various functions—

speaking, chewing, parafunctional habits, etc.

(Peck 2016).

From the simplest restorations to complex full-

mouth rehabilitations, this dynamic jaw move-

ment must be taken into consideration, otherwise

it could lead to the failure of the restoration(s).

While simple dental restorations (i.e.—ﬁllings)

are easily done directly in the mouth and, there-

fore, adjusted to this dynamic movement

intraorally, more complex restorations (i.e.—

crowns) are created indirectly on the patient’s

dental models. Traditional methods consisted of

these restorations being fabricated on stone dental

models of the patient’s upper and lower arches

which were mounted on a dental articulator

through the utilization of a facebow record.

A facebow (Fig. 5.5) is an intra-oral dental

device that records the position of the patient’s

arches and then translates that position to the

articulator. Articulators can range from simple to

fully adjustable models, but all share the same

limitation—they are mechanical devices, which

are limited in the range of motion they can mimic

because of the dynamic and non-linear movement

of the lower arch.

In today’s world, digital workﬂows for the

fabrication of dental prostheses can also generate

basic mandibular movement. This is accom-

plished by the movement of the surface scan, or

3-dimensional model, of the mandible in a direc-

tion (right, left, forward) along a ﬂat plane that

exists between the maxilla and mandible. When

the mandibular scan collides with the maxillary

5 The Impact of Technological Innovation on Dentistry 85

Fig. 5.5 Digital pathways of mandible being illustrated with virtual models in design software illustrated by the black dots

scan, the software will continue to move the scan

but make adjustments that eliminate the intersec-

tion of the scans. This movement mimics intra-

oral mandibular movement in that it replicates

tooth-guided movement. However, the generation

of this movement fails to consider the inﬂuence of

the neuro-musculature system. To counteract this

issue, specialized extraoral systems have been

developed that capture the dynamic motion of

the mandible (Kwon et al. 2019). These systems

vary in the way data is captured but are consistent

in that the dynamic motion is transferred into the

design software. This allows the software to

mimic the actual movement of the patient’s man-

dible, with all factors included. Deviations,

non-linear pathways, and other 3-dimensional

movements can be recreated within the software,

eliminating the solely mechanical movements

associated with mechanical articulators (Fig. 5.6).

86 R. Zimmermann and S. Seitz

This digital workﬂow is comprised of digital

versions of the facebow and articulator. In order

to capture the movement of the jaw, sensors are

attached to the patient’s head that act as a digital

facebow. The ﬁrst step is registering the maxilla

to the sensors, and then registering it to the

mandible.

Fig. 5.6 Sensors embedded in the cranial apparatus are

used to track sensors located in the mandibular apparatus,

which is attached to the patient’s teeth

After this is completed, the patient is instructed

to move their mandible through speciﬁc

positions, during which the sensors record the

3D movement pathway of the mandible. This

data is imported into speciﬁc software that allows

the user (either clinician or dental technician) to

accurately replicate the patient’s actual curvilin-

ear jaw motion with the patient’s mandibular

virtual model. This allows the user to evaluate

the mandibular path of movement as it relates to

maxillary arch and recognize “interferences”or

occurrences where the movement is limited,

between the teeth of the opposing dental arches.

This technology can be used in combination with

CBCT to diagnose temporomandibular joint dis-

order, or TMD (Liao 2021). Evaluation of the jaw

movements, along with the analysis of both the

shape and wear in the condyle and fossa of the

joint, can aid the dentist in diagnosing the causes

speciﬁc for each patient to determine the most

effective treatment for that individual. The ability

to record and analyze a patient’s actual mandibu-

lar movement is important in the evaluation of

temporomandibular jaw disorders and the ability

to track them over time. More research is cur-

rently needed in this area, but it indicates huge

potential for this ﬁeld of dentistry.

The digital applications described above are

being routinely done today for the creation of

dental restorations. Clinicians are also using

these 3-dimensional virtual models to better eval-

uate and discuss treatment options, with their

patients and other oral health care providers.

The intra-oral scan is the simplest and most

basic 3D model of a patient, and it is also the

foundation upon which other technologies can be

utilized.

A limitation that currently exists with digital

dental technology is the inability to create a

patient model that combines all of the various

digital scans into one virtual model that is able

to mimic the dynamic movement of the patient’s

extraoral soft tissue during mandibular movement

that is smile, or any other facial animation. This

type of virtual model would prove incredibly

beneﬁcial and would have the greatest impact in

the area of esthetic dentistry and smile design.

These two areas require the analysis of a patient’s

animated lip line to be able to achieve the optimal

esthetic outcomes for the patient.

5 The Impact of Technological Innovation on Dentistry 87

So far in this chapter, it has been explained

how a patient-speciﬁc model for dentistry is cre-

ated, including the advantages and disadvantages

of the pathways and the limitations of these vir-

tual models. The use of virtual models is easily

appreciated in reference to the fabrication of vari-

ous dental prostheses due to the fact that they are

a replacement for traditional stone models. How-

ever, there are numerous other ways in which

these virtual models are being used in general

dentistry and the various dental specialties that

warrant discussion in further detail.

5.4 Virtual Model Utilization

in Oral Maxillofacial Surgery

One of the areas that beneﬁts the most from these

virtual models is oral maxillofacial surgery,

which was ﬁrst discussed at the beginning of the

chapter. From orthognathic jaw surgery to

implant surgery, virtual models have become

mainstream in the diagnosis and treatment

planning for these surgeons (Otranto de Britto

Teixeira et al. 2020). Orthognathic surgery is

used to correct deformities in the upper or lower

jaw and the associated incorrect positioning of the

teeth. To do this effectively, the surgeon utilizes a

specialized software that allows the user to simu-

late various osteotomies (ex-sagittal split or verti-

cal ramus osteotomy) on the virtual model while

taking into consideration the patient’s speciﬁc

anatomical structures. For example, when

planning mandibular osteotomies with a virtual

model, the user is able to virtually track the infe-

rior alveolar canal (IAC), a canal that houses an

important neurovascular bundle, and creates a

mesh of the pathway as it goes through the length

of the lower jaw. This mesh provides the user a

3-dimenional view of the IAC pathway, allowing

for more precise planning to avoid the vital

structures within. This is incredibly important

because any damage to the inferior alveolar

nerve can cause symptoms including: numbness,

pain, burning or tingling in the lips, chin, and

gums; drooling; or impaired speech. In addition,

once the virtual osteotomies are performed, the

sections can be repositioned and allows for a

virtual generation of the model that depicts the

proposed surgical outcome.

In addition to creating a virtual rendering of

the ﬁnal surgical results of osteotomies, surgeons

are able to use certain specialized software to

predict the soft tissue and hard tissue outcomes

of orthognathic surgery. The software utilizes

databases of documented cases to predict the

soft tissue outcome based on the hard tissue

changes. It has been shown that the predicted

outcome for hard tissue changes is reasonably

accurate, within 1 mm of the actual surgical

outcomes. However, the predictions for soft tis-

sue outcomes are more varied with the greatest

area needing improvement being the region

around the lower lip.

5.5 Virtual Model Utilization

in Orthodontics

In the area of orthodontics, the use of lateral

cephalometric analysis via a 2D image more

widely used over CBCT due to the reduced

amount of radiation exposure to the patient. How-

ever, there are certain cases when the CBCT and

3D visualization are superior for diagnosis and

treatment planning. The primary advantage in 3D

visualization is due to the inherent issue with 2D

radiographic images; the fact that impacted teeth

are superimposed over the other anatomical

structures, and it is difﬁcult to ascertain the

exact position or impact on the tooth or

anatomical structure. The 3D models allow com-

plete visualization of these areas (Baan et al.

2020).

One of these cases involved the impaction of

maxillary canines, which are the second most

commonly impacted tooth, after third molars

(wisdom teeth). Impacted teeth are teeth in

which tissue, bone, or another tooth has prevented

it from reaching its normal position in the mouth.

The surgical exposure of these teeth is one of the

most common indications for CBCT imaging in

orthodontics. In this type of case, the virtual

model improves the treatment planning by

allowing visualization of the precise location in

the arch, including the proximity to adjacent teeth

and other vital structures. In addition, it allows for

a more accurate evaluation of the tooth follicle

and enables the assessment and extent of any

existing adjacent tooth resorption. This visualiza-

tion enables the orthodontist to better plan the

surgical access, orthodontic bracket placement,

and extrusion path for the impacted tooth, which

results in a better outcome for the patient.

88 R. Zimmermann and S. Seitz

Supernumerary teeth are extraneous teeth that

can develop anywhere in a person’s mouth. The

most common area for these teeth to be found is in

the anterior maxillae and they can be difﬁcult to

differentiate from normal dentition. These teeth

are usually impacted or unerupted. Virtual models

allow the orthodontist to precisely locate the teeth

and determine their true morphology. This is

important as it will aid in treatment planning and

help determine whether the teeth are retrievable

and able to be retained or if they need to be

extracted.

5.6 Virtual Model Utilization

in Endodontics

In dentistry, endodontics is the area that focuses

on the dental pulp, the roots of teeth, and the

tissue surrounding the roots. The majority of end-

odontics revolves around the treatment of the root

canals of teeth. Root canal morphology is a com-

plex three-dimensional structure that varies

between teeth and is even found to be distinct

within certain populations. Maxillary molars

present with numerous variations that challenge

even the most experienced endodontist. Tradi-

tional 2-dimensional radiographs are the most

widely used method to diagnose and treatment

plan endodontic therapy, however, these images

provide limited information and are inﬂuenced by

X-ray angulation, the overlapping of anatomical

structures, and contrast. For these reasons, the use

of CBCT has been shown to be incredibly useful

for complex cases.

The ability of a clinician to navigate through a

3D image of a tooth allows for better visualization

and, therefore, a better understanding of the root

canal morphology (Shah and Chong 2018).

Curves, accessory canals, and even fractures of

the root can be seen from different angles,

allowing the clinician to better diagnose, plan,

and provide treatment for the patient. It has also

been used to help differentiate between endodon-

tic and non-endodontic pathologies. Emerging

dental software allows the user to virtually plan

endodontic treatment by identifying anatomical

abnormalities, calculating exact measurements,

and choosing which instruments should be used

prior to the actual treatment. Currently, the main

drawback in using CBCT and virtual modeling

for endodontics lies with the amount of radiation

exposure, which is higher than the conventional

2D radiographs. For this reason, the use of CBCT

in endodontics is only recommended for complex

cases at this time.

5.7 Virtual Model Utilization

in Implant Dentistry

Dental implantology involves the combination

two different areas of dentistry—surgery and

prosthetics—to provide treatment for a patient.

This treatment can range from a simple, single-

tooth replacement to full arch reconstruction

supported by numerous dental implants. For any

case, accurate placement is essential to achieve

optimal esthetic and functional prosthetic

outcomes. However, patient anatomy may limit

the proposed location of the implant, requiring

additional planning or even a completely different

approach (Kernen et al. 2020).

The ﬁrst step in digital treatment planning of a

dental implant is the acquisition of surface scans

of the patient. For dentate patients, this is accom-

plished with the use of an intra-oral scanner, as

discussed previously. Edentulous patients,

however, are more complex as soft tissue can be

more challenging to capture and does not exhibit

adeﬁnitive shape like hard tissue due to its mobil-

ity. For this reason, there are distinctive and dif-

ferent pathways to create virtual models for

dentulous and edentulous patients.

5 The Impact of Technological Innovation on Dentistry 89

For edentulous patients, a well-ﬁtting remov-

able prosthesis (denture) is required—one that is

well adapted to the patient’s soft tissue. At the

CBCT scanning appointment, ﬁduncles

(specialized small metal spheres for use in

CBCT). are attached to the patient’s current pros-

thesis. The patient is then scanned with the

modiﬁed prosthesis in place. Subsequently, the

prosthesis itself is scanned separately while sit-

ting on a CBCT table. Afterwards, the CBCT

scan of the patient wearing the prosthesis and

the prosthesis alone are imported into a speciﬁc

dental implant planning software that utilizes arti-

ﬁcial intelligence to align the patient CBCT to the

prosthesis CBCT using the ﬁduncles. The soft-

ware will take the prosthesis CBCT scan data and

create a virtual replica (mesh) of the prosthesis.

The position of the prosthesis to the patient’s hard

tissue is accomplished by the alignment of the

ﬁduncles. The next step in the software is to

create a virtual mesh of the soft tissue by reverse

engineering the tissue side of the prosthesis. Once

completed, a virtual model of the patient’s bone

with overlying tissue mesh is formed (Fig. 5.7).

For both edentulous and dentate patients,

the next step after merging of the surface scans

to the CBCT ﬁle comes the actual planning of the

implant case.

Dental implants come in a wide range of

lengths, diameters, and shapes. Historically,

2-dimensional radiographs (panographs), along

with acetate overlays depicting different implant

sizes, were used to plan for surgical placement.

Surgical guides were then fabricated on stone

models that had a diagnostic wax-up of the pro-

posed ﬁnal restorations. The main limiting factor

with this method was the inability to take the

patient-speciﬁc anatomy into consideration

when planning. While some anatomical structures

are visible on 2D radiographic ﬁlm (ex-inferior

alveolar nerve), others are hidden due to

overlapping of other anatomical structures

(ex-palatal foramen).

For dentate patients, a proposed design of the

prosthesis replacing the missing teeth must be

created. By doing this, the restorative doctor is

conveying to the surgeon the ideal position of the

implant based on the patient’s existing dentition.

This proposal takes into account a number of

factors including the placement of the ﬁnal resto-

ration with respect to adjacent teeth and opposing

dentition. The goal is to place dental implants in

positions that will support ﬁnal restorations that

are in harmony with the patients existing denti-

tion and maxillomandibular relationships. Failure

to do this results in a signiﬁcantly higher proba-

bility of a broken prosthesis and/or failed implant.

Current dental technology supports the virtual

design of the patient’sﬁnal prosthesis by

allowing the restoring dentist to create a virtual

prosthesis that takes all of these important factors

into consideration and ensures the ﬁnal prosthesis

is in harmony with the patient’s dentition. Once

completed, the design is integrated into the sur-

face scan and aligned with the patient’s CBCT.

Afterwards, the surgeon can plan and place the

implant in the optimal location using the pro-

posed prosthesis as a reference.

Once the planning for both the prosthesis and

the implant is complete, either in dentate or eden-

tulous patients, a surgical guide can be created

based on the virtual implant plan. Surgical guides

are aids that help the surgeon orient the implant

during the implant surgery to achieve the pro-

posed correct location, depth, and angulation

needed for the prosthesis. Surgical guides can be

utilized in simple, straightforward cases requiring

the replacement of some teeth with implants, or in

more complex cases. Complex cases require more

advanced guides, usually multiple guides for a

single case, which require advanced virtual

modeling techniques to create.

In certain implant rehabilitation cases, an

alveoloplasty (bone reduction) is needed to create

restorative space for the ﬁnal prosthesis. The

workﬂow is similar to the ones already stated,

however, during the implant planning phase, the

amount of bone needing to be removed can be

determined based on the restorative components.

90 R. Zimmermann and S. Seitz

Fig. 5.7 Patient intra-oral scan aligned to patient CBCT for implant planning

Once the surgeon virtually arranges the implant

along with the restorative components in place,

the amount of bone needed to be removed can be

measured on the virtual model. Once that amount

is determined, the software will allow the user to

remove and recontour the bone, creating a virtual

bone reduction that gives the surgeon a guide for

what has to be done to accomplish the

alveoloplasty. This workﬂow is best undertaken

by experienced clinicians who have a thorough

understanding of bone physiology and

remodeling and who can predict the outcome of

the bone reduction surgery. Once this workﬂow is

completed, the complex surgical guide can be

designed. The original, unaltered virtual model

is used to create the ﬁrst part of the guide, while

the modiﬁed model that has undergone the virtual

alveoloplasty, is utilized to create the bone reduc-

tion and implant placement. While there are

limitations to these complex surgical guides, it

has been shown that the use of these guides

decreases surgical time and improves prosthetic

outcomes, which warrants the time spent

creating them.

5 The Impact of Technological Innovation on Dentistry 91

It is not uncommon for some type of bone

grafting procedure to be necessary in conjunction

with implant placement. While it is difﬁcult to

determine the real-world outcome of virtual

grafting due to various factors, such as patient

health and behavior, virtual modeling allows the

surgeon to predict if grafting is going to be

needed and the amount of graft material that

should be used. It is well established that at least

1.5 mm of bone is needed to surround a dental

implant for successful integration. If it is deter-

mined that the proposed implant location lacks

the 1.5 mm of bone in an area while virtually

planning the implant, the surgeon can plan for

the placement of a bone graft. There are various

types of bone that can be utilized for a bone graft

and the amount of bone grafting needed aids the

surgeon in determining which of these materials

will be the best ﬁt for the speciﬁc situation.

An additional area that commonly requires

bone grafting is the maxillary sinus region.

Often, the sinus is located too low for the desired

implant to be completely encased in bone, so

additional bone is placed in the bottom of the

sinus to ensure the implant is fully in bone and

does not protrude into the sinus. It is not uncom-

mon for a sinus lift to be performed at the same

time of the implant placement. This procedure

can also beneﬁt from virtual planning for numer-

ous reasons.

The most common approach for a sinus lift is

to access the sinus through a lateral window that

is surgically cut into the lateral wall of the sinus

and the sinus membrane is carefully elevated

from the ﬂoor of the sinus. Once the access is

made, grafting material is then placed below the

sinus membrane and the bone window is

replaced. During the planning phase, a virtual

model can illustrate the position of any septa or

deviation in the internal area of the sinus that

could complicate the surgical approach. More

importantly, the model allows for the visual track-

ing of the posterior superior alveolar artery and

nerve to determine the best approach to avoid

injury. Some implant planning software will

even allow the user to determine a specialized

model of the sinus and calculate the amount of

bone grafting material needed to accomplish the

sinus lift (Fig. 5.8).

This aids in eliminating the unnecessary waste

of bone grafting material by providing the amount

needed. An alternative approach to accessing the

sinus is through the residual crestal ridge of the

jaw. However, for this approach to be successful,

certain anatomical features must be present. For

example, the area must be free of any septa and

have a certain amount of bone thickness

remaining. If not, then the lateral window

approach is recommended. While there has been

many of these surgeries accomplished before

patient-speciﬁc modeling, this technology has

helped to better prepare the surgeon and decrease

the surgical time.

Examples of some cases are provided below:

5.7.1 Case Example #1

A patient presented with a removable appliance

(“ﬂipper”) replacing her four anterior teeth. The

patient was happy with the existing esthetics of

the appliance, including the tooth shape and

position of the teeth, but did not like the poor ﬁt

and the fact that it was removable and not a ﬁxed

prosthesis (Fig. 5.9).

92 R. Zimmermann and S. Seitz

Fig. 5.8 The yellow areas outline the patient sinus in this implant planning software which aid in planning of implants in

this region

The patient was interested in obtaining a more

permanent ﬁxed restoration that could mimic her

natural teeth, so she was referred for implant

therapy.

Fig. 5.9 Intra-oral picture of patient wearing their remov-

able appliance

Initially, a traditional panogragh was taken and

a clinical examination of the patient’s maxillary

alveolar ridge was performed. Both the

panogragh and the clinical examination revealed

that the patient had an adequate alveolar height

and the necessary ridge width for the placement

of implants (Fig. 5.10).

The standard of care at this clinic required the

dentist to obtain a CBCT for all patients receiving

dental implant therapy, so the patient was referred

Fig. 5.10 Intra-oral picture of patient without the remov-

able appliance

to the Radiology clinic for the CBCT and

subsequent radiology interpretation. Intra-oral

scans were also taken of the patient’s maxillary

and mandibular arches, along with her existing

removable partial appliance to be used as a diag-

nostic wax-up for the ﬁnal prosthesis.

5 The Impact of Technological Innovation on Dentistry 93

Fig. 5.11 Virtual model of

patient rendered

from CBCT

Upon interpretation of the CBCT, it was

determined that the alveolar ridge had more

buccal-lingual resorption than what was

initially observed, with alveolar defects due to

previous extractions. It also revealed thin and

immature bone, as opposed to the more solid

buccal cortical plate (Fig. 5.11).

When placing implants, the density and vol-

ume of existing bone are critical for successful

osseointegration. If the existing bone is not ideal,

bone grafting or more advanced surgical

procedures are required. In this particular case, a

virtual model of the patient was created to better

visualize the situation that revealed the true extent

of the bone loss in the anterior region. While

virtual models from CBCT’s are not 100% accu-

rate, they can provide the clinician with a clearer

picture of the clinical situation. Given the anterior

location of the planned dental implants, the

patient’s concern for the most esthetic outcome,

and the amount of bone loss observed, it was

decided to virtually plan the implant case.

The ﬁrst step was the creation of the virtual

model from the patient’s CBCT. For this case, the

teeth were segmented in the maxillary bone to

better visualize the trajectory of the roots of the

canines (Fig. 5.12).

When segmenting different areas of the model,

it has been found that the ability to assign a

different color to each area and adjust the trans-

parency of each segment are useful tools for the

dentist. These tools allow for better visualization

of the different areas, including bone, roots of the

teeth, etc. It is particularly useful when overlaying

various surface scans that contain multiple

segmentations.

After the CBCT model was completed, the

surface scans (maxillary and mandibular) were

then aligned to it, followed by the scan of the

interim appliance (Fig. 5.13).

For this case, a virtual scan of the face would

have been useful given the importance placed on

esthetics. However, due to the fact that the patient

was happy with the esthetics of the current

removable appliance, it was decided to import a

scan of it to aid in planning. At this point, a

complete virtual model of the patient had been

created within the dental implant planning

software.

The replacement of anterior teeth with dental

implants can be esthetically challenging, espe-

cially with multiple missing teeth complicated

by bone loss. In this case, it had to be determined

whether two implants and a bridge or four

implants with separate crowns would be used to

replace the missing teeth. There are advantages

and disadvantages associated with both, however,

the existing bone architecture and the possibility

of necessary additional procedures must be con-

sidered to achieve the desired outcome.

94 R. Zimmermann and S. Seitz

Fig. 5.12 Virtual model of patient showing segmentation of teeth/roots and proposed implant locations

Fig. 5.13 Virtual model of implant planning showing proposed position of implants (yellow) to the patient’s removable

appliance (light blue)

Once the full virtual model was completed and

the virtual planning of the case had begun, it was

realized that the width of the remaining ridge was

insufﬁcient for the placement of four dental

implants without requiring a large bone graft. It

was also determined that the placement of just

two dental implants at certain positions would

only require a minimal grafting procedure.

5 The Impact of Technological Innovation on Dentistry 95

The ability to place implants in the virtual

model also provides visualization of the neces-

sary implant trajectory. This is important in the

restorative phase of implant therapy as it will

dictate the type of restoration that can be placed.

Therefore, when planning an implant, the desired

restorative plan must be balanced with the avail-

able bone to achieve optimal outcomes. This is

where the scan of the patient’s existing prosthesis

becomes important. By overlaying her appliance

in the virtual model, the clinician can visualize the

effect that the trajectory of the implant will have

on the ﬁnal prosthesis. In this particular case, it

was determined that the preferred method of res-

toration would not be feasible since the trajectory

emerged from the incisal, or bottom edge of the

teeth. The decision had to be made either to

change the restorative design or perform larger

and more complex grafting techniques. After con-

sulting with the patient and informing her of the

possibilities, she elected not to undergo additional

large surgical procedures and the restorative plan

was changed to accommodate the necessary

implant trajectory. Once the implant size and

position were ﬁnalized, a surgical guide was

fabricated to facilitate the planned placement of

the implants into the proposed positions. As with

all implant surgeries, the surgical guide was

fabricated using additive technology

(3D printing) and a specialized biocompatible

sterilizable resin was utilized.

At the time of surgery, a full-thickness perios-

teal ﬂap was created and reﬂected, which allowed

complete visualization of the alveolar ridge. In

the model, the center point of the ridge appeared

rough and thin—this is usually indicative of thin,

immature bone. While in the photograph the bone

appears to be solid clinically, it was thin and

porous (Fig. 5.14).

As predicted by the virtual model, a small

grafting procedure was needed.

Fig. 5.14 Intra-oral picture showing osseous architecture

after reﬂection of soft tissue

5.7.2 Case Example #2

When performing prosthetic reconstructions of

patients’dentitions, one must take the mandibular

movement into account, otherwise the

restorations will fail, or the patient will generate

temporomandibular symptoms. In traditional ana-

log workﬂows, the ﬁrst step is to obtain a

maxillomandibular relationship utilizing a

facebow. Facebow complexity ranges from sim-

ple to fully adjustable, with the fully adjustable

being the most complex and requiring hours for

an experienced clinician to use. The facebow is

then used to transfer the position of the maxillary

arch to an articulator to represent the patient’s

temporomandibular joint and both upper and

lower arches. The movement allowed by the artic-

ulator depends on the model used—a simple

articulator only allows for an open/close move-

ment, while the fully adjustable models allow for

more complex movements which more accurately

mimic the patient’s jaw movement. However, it is

still a mechanical device that is limited by the

features and build—namely the moving parts are

restricted to ﬂat rails. Therefore, the natural

movement of the mandible is not accurately

reﬂected. For example, when a patient open and

closes, the mandible may not move in a straight

vertical line but has a deviation to either side

during the movement. This deviation could be the

result of the person’s natural joint, previous

trauma to the joint, or a result of temporomandib-

ular disorder. Due to the lack of movement accu-

racy, the provider will need to perform chairside

adjustments in order to get the prosthesis in har-

mony with the patient’s oral complex. The

amount of time required for these adjustments is

related to the extent/size of the restoration being

done. A single tooth may be ﬁve to ten minutes,

while a full-mouth rehabilitation can take several

hours. Additionally, the more accurately the artic-

ulator mimics the natural movement of the

patient, the less time is spent in adjustments at

the time of delivery.

96 R. Zimmermann and S. Seitz

As mentioned earlier, digital technology that

allows for the capture of a patient’s mandibular

movement can be used with surface scans to

create an accurate virtual model of a patient’s

orthognathic system. This model can be used for

numerous diagnostic and therapeutic treatments,

but its biggest advantage lies with the develop-

ment of patient-speciﬁc restorations. To create

this type of virtual model, intra-oral scans of the

maxillary and mandibular arches are taken. The

next step is to capture the mandibular movement.

To do this, specialized headgear containing infra-

red cameras is placed on the patient. A sensor is

then used with specialized bite plates to register

the position of the maxillae and then the mandi-

ble. At this point, the same information from the

traditional facebow has been obtained.

The next step in this digital process involves

the attachment of the sensor to the mandibular

biteplate and having the patient perform jaw

movements in a speciﬁc order. As the patient

goes through the motions, the infrared cameras

in the headset capture the 3-dimensional move-

ment of the sensor on the biteplate. The software

then creates a demonstration of all mandibular

movements performed by the patient. At the

same time, the software creates tracings of the

patient’s condyles that can be used for diagnosis

(Fig. 5.15). These tracing can be used in identiﬁ-

cation of pathologies or unusual jaw movement,

as discussed previously.

Once the movements are tracked, the intra-oral

scans are imported into dental-speciﬁc Computer-

aided design (CAD) software along with the algo-

rithm from the digital facebow. The algorithm

orients the surface scans onto a virtual articulator,

creating a virtual setup resembling the traditional

analog articulator.

The CAD software can replicate the patient’s

mandibular movement without any limitations.

The user is able to see any deviations from normal

and the software creates visual aids by displaying

a series of dotted pathways the mandible goes

through during functional movements. Speciﬁc

movements (example—left lateral movement),

may be individually selected and visualized.

Since these movements are in the virtual world,

they will reﬂect any deviation that the patient may

have in reality.

The software is also capable of generating

meshes of individual movement pathways,

which prove incredibly useful for the develop-

ment of prostheses by eliminating various

interferences that would have been present if

only a static relationship was used.

Interferences arise when tooth cusp tips hit

opposing teeth prematurely or out of harmony

with the other parts of the orthognathic system.

These interferences were referenced earlier and

are the cause of necessary chairside adjustments

that extend the time the patient must be in the

chair. When activated, the CAD software will

utilize the dynamic jaw movements to create a

restoration that will function most effectively for

the patient (Fig. 5.16).

These pictures illustrate such a case. This

patient needed two full coverage posterior

restorations. For various reasons, the most poste-

rior tooth in an arch is a little more challenging to

develop a proper occlusion when restoring. For

these restorations, the digital facebow was taken,

along with intra-oral scans of the patient. The

virtual patient was created as described above.

During the design process, the user was able to

visualize and toggle among the various occlusal

contacts on both the existing dentition and the

proposed prostheses to determine the best occlu-

sion for the ﬁnal restorations. In addition, the user

was also able to move the virtual mandible

through various motions to help better visualize

the mandibular pathway and possible

interferences.

5 The Impact of Technological Innovation on Dentistry 97

-4 -2 0246810 12 14 16 16 14 12 10 86420-2 -4

-8

-6

-4

-2

-8

-6

-4

-2

Sag. Condyle Protrusion, left

Sag. Condyle Protrusion, right

Hor. Condyle Laterotrusion, right Hor. Condyle Laterotrusion, left

Bennett

Retrusion RetrusionShift Angle Shift Angle

ISS ISS Bennett

mmmmmmmm

mmmmmm

-1

-2

-3

-4

-5

-6

-7

-8

-9

-10

-11

-5-1.50543210-1-2 1 -1 -0.5 00.5

-3.5

-3

-2.5

-2

-1.5

-1

-0.5

-3.5

-3

-2.5

-2

-1.5

-1

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3210-1-2-32310-1-2-32310-1-2-32310-1-2-3

-4 -3 -2 -1 012

Fig. 5.15 Condylar pathway tracings obtained from mandibular tracking

The software is able to illustrate the patient’s

occlusion utilizing a heatmap to demonstrate the

degree of contact. Red and yellow colors indicate

intersection of surfaces, while the blues indicate

areas of contact—light blue indicates a heavy

contact while dark blue indicates light contact

between teeth. When the restorations are ﬁrst

generated by the software, they have red and

yellow spots indicating they are actually

intersecting with the opposing surface scan

(Fig. 5.17).

Using the adapt function, the software adjusted

the proposed design of the restoration to be in

harmony with the rest of the patient’s dentition

(Fig. 5.18).

During the try-in of the two restorations, the

patient was instructed to go through all the vari-

ous mandibular movements while marking paper

was placed between the restorations and opposing

dentition. One can see how close the markings on

the ﬁnal restorations align with those indicated in

the design software (Fig. 5.19).

This demonstrates the accuracy involved in

how the digital facebow translates both

98 R. Zimmermann and S. Seitz

Fig. 5.16 Virtual models in lateral excursion with heat map visible (blue/light blue on teeth) illustrating interfering points

5 The Impact of Technological Innovation on Dentistry 99

Fig. 5.17 Virtual model of patient with proposed crowns (yellow) design showing interferences via heat map

100 R. Zimmermann and S. Seitz

Fig. 5.18 Proposed crown design after adjustment for interferences

maxillomandibular relationships and mandibular

movement. While this case illustrates the use of

virtual models and workﬂows to increase the

accuracy of restorations, one can easily appreciate

the beneﬁt of using this technology on larger

cases as well.

5 The Impact of Technological Innovation on Dentistry 101

Fig. 5.19 intra-oral picture of crowns at initial try-in

5.8 Conclusion

The exponential rate at which digital technology

is advancing in dentistry is apparent in the discus-

sion in this chapter. Consequently, the outcomes

for both dentists and patients are constantly

improving. New developments in both hardware

and software are allowing for better diagnosing,

planning, and treatment with the creation and

utilization of virtual models and virtual patients.

Dental procedures will be associated with less

treatment timeframe and fewer complications for

the patients, along with many other advantages,

due to these advances. However, based on recent

technological developments, it seems the best is

yet to come.

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s00784-018-2338-9

Advanced 3D Visualization and 3D

Printing in Radiology 6

Shabnam Fidvi, Justin Holder, Hong Li, Gregory J. Parnes,

Stephanie B. Shamir, and Nicole Wake

Abstract

Since the discovery of X-rays in 1895, medical

imaging systems have played a crucial role in

medicine by permitting the visualization of

internal structures and understanding the func-

tion of organ systems. Traditional imaging

modalities including Computed Tomography

(CT), Magnetic Resonance Imaging (MRI)

and Ultrasound (US) present ﬁxed

two-dimensional (2D) images which are difﬁ-

cult to conceptualize complex anatomy.

Advanced volumetric medical imaging allows

for three-dimensional (3D) image post-

processing and image segmentation to be

performed, enabling the creation of 3D volume

renderings and enhanced visualization of per-

tinent anatomic structures in 3D. Furthermore,

3D imaging is used to generate 3D printed

models and extended reality (augmented real-

ity and virtual reality) models. A 3D image

translates medical imaging information into a

visual story rendering complex data and

abstract ideas into an easily understood and

tangible concept. Clinicians use 3D models to

comprehend complex anatomical structures

and to plan and guide surgical interventions

more precisely. This chapter will review the

volumetric radiological techniques that are

commonly utilized for advanced 3D visualiza-

tion. It will also provide examples of 3D print-

ing and extended reality technology

applications in radiology and describe the pos-

itive impact of advanced radiological image

visualization on patient care.

S. Fidvi (✉) · J. Holder · S. B. Shamir

Department of Radiology, Monteﬁore Medical Center,

Bronx, NY, USA

e-mail: sﬁdvi@monteﬁore.org;juholder@monteﬁore.org;

sshamir@monteﬁore.org

H. Li · G. J. Parnes

Department of Radiology, Jacobi Medical Center, Bronx,

NY, USA

e-mail: lih18@nychhc.org;parnesg1@nychhc.org

N. Wake

GE Healthcare, Aurora, OH, USA

Center for Advanced Imaging Innovation and Research,

NYU Langone Health, New York, NY, USA

e-mail: Nicole.Wake@ge.com

#The Author(s), under exclusive license to Springer Nature Switzerland AG 2023

E. Abdel Meguid et al. (eds.), Biomedical Visualisation, Advances in Experimental Medicine and

Biology 1406, https://doi.org/10.1007/978-3-031-26462-7_6

103

Keywords

Radiology · Computed Tomography (CT) ·

Magnetic Resonance Imaging (MRI) and

Ultrasound (US) · 3D visualization · 3D

printing · Extended reality technology

6.1 Introduction

Medical imaging is of vital importance in

depicting the internal visualization of organ

systems and their function. Computed Tomogra-

phy (CT), Magnetic Resonance Imaging (MRI)

and Ultrasound (US) are the core modalities used

in biomedical imaging that display

two-dimensional (2D) images in ﬁxed

orientations. Advanced volumetric medical imag-

ing expands upon this 2D imaging by creating

three-dimensional (3D) images to be displayed on

the conventional 2D imaging screens which are

viewed by physicians, patients, and their families

alike. 3D processing techniques facilitate diagno-

sis and enable quantitative analysis of the

acquired images.

104 S. Fidvi et al.

3D printing, augmented reality (AR), and vir-

tual reality (VR) are 3D imaging methods used to

create anatomic models. Creating 3D models

from radiological imaging data is a complex pro-

cess that involves manipulating the original 2D

images into a volume that represents patient-

speciﬁc anatomy. 3D anatomic model creation

includes image acquisition, image segmentation,

and computer-aided design (CAD) modeling. The

manipulated data is then optimized for the

advanced visualization method of choice—3D

printing, AR, or VR. All forms of 3D modeling

transform biomedical information into a visual

display that renders abstract and complex

diseases readily comprehensible. 3D modeling

from medical imaging data elucidates complex

anatomic structures and highlights pathology

which enables clinicians to plan surgical

procedures more effectively and enhance patient

outcomes. For example, 3D models are used to

plan tumor resections within the abdomen, pelvis,

breast, and musculoskeletal systems, as well as to

facilitate many cardiac and neurointerventional

procedures. In addition to clinical applications,

3D models have been used to improve trainee

and patient education.

3D modeling has been used in medical educa-

tion for generations to demonstrate both normal

anatomy and complex pathologies, and many

medical student and radiology residency training

programs implement 3D printing and extended

reality technologies (Burns et al. 2022).

Institutions also use these models to assist

patients and their families in understanding medi-

cal conditions, surgical goals and surgical

approaches. Patient speciﬁc, anatomically accu-

rate 3D models enhance patient education and the

consent process by helping the patient and their

family to better understand their pathology as

well as the surgery that will be performed and

the inherent risks the procedure entails

(Eisenmenger et al. 2017; Tzavellas et al. 2020).

This leads to more effective communication, ele-

vated patient satisfaction, and conﬁdence in

selecting a treatment plan (Mitsouras et al. 2015).

This chapter provides an overview of the

beneﬁts and limitations of the traditional imaging

modalities of CT, MRI, and Ultrasound. Further-

more, the chapter highlights the volumetric radio-

logical techniques that are commonly utilized for

advanced 3D visualization and provides

examples of 3D printing and extended reality

technology applications in radiology. The chapter

discusses examples that demonstrate how

advanced medical imaging techniques facilitate

myriad cardiovascular and neurointerventional

procedures. Additionally, the chapter focuses on

how the use of 3D imaging can be used to plan

tumor resections in the abdomen, pelvis, breast,

and musculoskeletal system. In summary, the

chapter highlights the tremendous positive impact

of advanced radiological image visualization

techniques like 3D printing in revolutionizing

patient care.

6.2 Medical Imaging Technologies

Imaging modalities including CT, MRI, and ultra-

sound are often used for advanced 3D image

visualization or to create 3D printed and extended

reality models (Wake et al. 2022). There are many

texts that describe these imaging modalities in

detail (Seutens 2009; Kalendar 2011; Brown

et al. 2014; Zagzebski 1996). Herein, the basics

are described in order to provide a background for

the 3D imaging and visualization methods com-

monly used in radiology and medicine.

Computerized Tomography is used most often

for advanced 3D image visualization and

modeling due to the ease of post-processing CT

data. A CT scan involves a series of X-rays deliv-

ered through a patient that is subsequently

detected by the scanner detector after passing

through the body. The quantity of X-rays

absorbed by the body determines the number of

X-rays that reach the CT detector, which in turn

utilizes software to assign grayscale levels

deﬁned as Hounsﬁeld Units (HU) to various

tissues. Tissues that do not absorb many X-rays

appear black, such as fat and air, while those that

absorb these rays appear white, such as bone and

intravenous contrast. Most organs and vascular

structures fall somewhere in between, as varying

degrees of gray. CT is thus able to distinguish and

isolate pathological processes from the back-

ground tissue based on the differing levels of

white, gray, and black. The radiologist deﬁnes

and separates objects on the acquired images to

generate 3D models, and the pathology must be

demarcated from the background tissue through

the post-processing step. This modeling process

thus works most effectively if the organ can be

easily separated from its environment, and if the

pathology can be accurately separated from the

organ. CT is conducive to this as there is a solitary

color scale to evaluate and the images are often

acquired in a standard, axial plane (Mitsouras

et al. 2015).

6 Advanced 3D Visualization and 3D Printing in Radiology 105

Magnetic Resonance Imaging is a more com-

plex imaging modality that provides good tissue

contrast without ionizing radiation. MRI uses

varying electromagnetic ﬁelds applied to a patient

in multiple conﬁgurations and directions to gen-

erate images in unlimited planes. There are con-

sequently many sequences available (e.g., T1, T2,

proton density, diffusion, dynamic contrast

enhanced) that highlight different tissues in vari-

ous ways and in different orientations. The signal

of tissue can have multiple different colors, or

intensities, depending on the pathology and

sequence used for display. MRI is more difﬁcult

to use than CT for 3D image visualization and 3D

printing. MRI has some limitations in

distinguishing pathology from background tissue

because it has thicker slabs than CT which results

in more structural overlap and impairs the

radiologist’s ability to diagnose with precision.

Finally, MRI is particularly susceptible to

artifacts that can distort images, which renders

contouring and isolating structures challenging

(Graves and Mitchell 2013; Morelli et al. 2011;

Bernstein et al. 2006). Setting these challenges

aside, what makes MRI so effective in radiology

is its excellent soft tissue contrast and the wealth

of information content in MR images.

Ultrasound is commonly used for advanced

3D imaging visualization. This imaging modality

employs high-frequency sound waves that spread

through the body, bounce off deep tissues, and

return to the machine to be processed (Prince and

Links 2006). The generated image is determined

by the different speeds and directions of

soundwaves emitted from the various tissues

and structures detected by the machine. Although

ultrasound is excellent for 3D image visualiza-

tion, it poses a challenge for 3D modeling as

ultrasound images are not obtained in the stan-

dard transverse, coronal, and sagittal orientations.

However, the use of specialized probes and image

processing software enhance the ability to per-

form a 3D ultrasound (Sepulveda et al. 2012).

This has allowed the progressive incorporation

of 3D-US into the routine fetal anatomical survey

to allow evaluation of select fetal anatomical

landmarks and serve as a complement to evaluate

detected fetal anomalies (Sepulveda et al. 2012).

The other limitation of ultrasound is that the

image appearance is entirely dependent on man-

ual transducer positioning and there is high

variability in organ and pathology orientations

and appearances (Wake et al. 2022).

6.3 3D Image Visualization

Volumetric medical images can be visualized on a

2D screen or a 3D display which effectively

represents the spatial relationships between adja-

cent structures. In order to properly visualize ana-

tomic structures in 3D or to create accurate

anatomic 3D models from medical imaging data,

the slice thickness must be sufﬁciently narrow

(e.g., 1 mm or less). If the slice thickness is too

large, then the 3D anatomical region of interest

will be distorted. For CT and MRI, 3D

acquisitions with isotropic resolution (e.g., 1 ×

1×1 mm) permit visualization of the anatomy

from any orientation, enabling proper depiction of

any pertinent anatomic structure.

Volume rendering is an important data visual-

ization technique used to create a 3D

representation of volumetric medical imaging

data, typically CT. Direct volume rendering

methods generate 3D representations of the vol-

ume data directly (Levoy 1988). An optical model

is applied to map data values to optical properties

such as color and opacity (Max 1995).

106 S. Fidvi et al.

Fundamental CT algorithms include Maxi-

mum Intensity Projection (MIP), Minimum Inten-

sity Projection (MinIP), and Shaded Surface

Volume Renderings (SS-VRT). MIP images are

obtained by projecting the voxel with the highest

attenuation value on every view throughout the

image volume, whereas MinIP images display

only the lowest attenuation value encountered in

a speciﬁc volume. MIP images are useful for the

detection of small lung nodules and MinIP

images are particularly useful for analyzing the

biliary tree and pancreatic duct. SS-VRT allows

for 3D visualization from volumetric data by

representing object surfaces (Udupa et al. 1991;

Hong and Freeny 1999; Fishman et al. 2006).

SS-VRT, which is particularly useful for

visualizing bone or vasculature, provides a 3D

perspective of that speciﬁc tissue. An important

variation of SS-VRT is virtual endoscopy

wherein the surface of the gastrointestinal tract

lumen, such as the colon, is displayed so that

lesions and polyps are readily detected. There

are thus multiple post-processing algorithms that

can be applied to 2D images to generate clinically

useful 3D images.

6.4 Image Segmentation

Image segmentation, the process of subdividing

an image into its constituent regions or objects, is

a highly relevant task in medical image analysis

and is important for many clinical applications.

Segmentation algorithms for grayscale images are

typically based on one of two basic categories,

both of which deal with properties of intensity

values: (1) discontinuity (partitioning an image

based on abrupt changes in gray level) and

(2) similarity (partitioning an image into regions

that are similar). Common segmentation

techniques available in most medical image

post-processing platforms include thresholding,

edge detection, and region growing.

Thresholding is the simplest method of image

segmentation and can be used to generate binary

images from a volumetric grayscale image

dataset. To differentiate pixels of interest, a com-

parison of each pixel intensity value with respect

to a threshold is performed. If the pixel’s intensity

is higher than the threshold, the pixel is set to one

value, i.e., white, and if it is lower than the thresh-

old, it is set a second value, i.e., black (Fig. 6.1).

Edge detection is a technique that ﬁnds the

boundaries of objects (edges) within images.

Edge detection works by detecting discontinuities

in brightness between adjacent structures and is

used most frequently for segmenting images

based on sudden local changes in intensity.

Region growing is a simple region-based

image segmentation method. It is classiﬁed as a

pixel-based image segmentation method since it

involves the selection of initial seed points. Once

a seed point or set of points has been identiﬁed, a

region is grown by appending to each set of

neighboring pixels that have predeﬁned

properties similar to the seed.

In the USA, when medical image post-

processing software is marketed for patient care

(i.e., Clinical Care or Diagnostic Use), the soft-

ware needs to have Food and Drug Administra-

tion (FDA) clearance. FDA-cleared software is

not required for research in the US. Outside the

USA, regulations vary. Table 6.1 highlights some

of the software products available for medical

image post-processing.

6.5 3D Printing

3D printing and extended reality technologies are

increasingly being used in medicine for a wide

range of applications including pre-surgical

planning, simulations, intraoperative guidance,

trainee education, and patient education. 3D

printing technologies can also be used to create

patient-speciﬁc surgical guides, implants, and

prosthetics.

Also known as additive manufacturing or

rapid prototyping, 3D printing uses an additive

6 Advanced 3D Visualization and 3D Printing in Radiology 107

Fig. 6.1 Example of bone thresholding from CT data using HU 226–1085. (a) Coronal view, (b) axial view, (c) sagittal view, (d) threshold settings, (e) 3D visualization of

segmented bone. Images courtesy of Nicole Wake, PhD, GE Healthcare

Product Company Website

method to create a 3D physical object layer by

layer. First patented in 1986 by Chuck Hull, 3D

printing technologies have quickly developed

over the years (Hull 1986). Although all 3D

printers use the basic additive fabrication method

that involves building the part one layer at a time,

they differ on the material types and techniques

used. Today, according to the American Society

for Testing Materials, there are seven major 3D

printing technologies (ISO 2021). These

technologies include binder jetting, direct energy

deposition, material extrusion, material jetting,

powder bed fusion, sheet lamination and vat

photopolymerization (Table 6.2).

108 S. Fidvi et al.

Table 6.1 3D visualization and segmentation programs utilized in radiology

FDA

Clearance

3D Slicer Brigham and

Women’s

Hospital

www.slicer.org None

Amira Thermo Fisher

Scientiﬁc

https://www.thermoﬁsher.com/us/en/home/electron-

microscopy/products/software-em-3d-vis/amira-software.html

Visualization

Analyze/

Analyze Pro

Analyze Direct www.analyzedirect.com None

AnatomicsRx Anatomics www.anatomicsrx.com None

AW Server GE www.gehealthcare.com 3D

Visualization

D2P 3D Systems www.3Dsystems.com 3D Printing

Dolphin 3D

Surgery

Dolphin/Patterson

Dental

www.dolphinimaging.com 3D

Visualization

F.A.S.T Fovia www.fovia.com 3D

Visualization

IBM

iConnect

IBM https://www.ibm.com/products/iconnect-access 3D

Visualization

iNtuition TeraRecon www.terarecon.com 3D

Visualization

Itk-SNAP University of

Utah & UPENN

www.itksnap.org None

MeVisLab Mevis www.mevislab.de None

Mimics Materialise www.materialise.com 3D Printing

Mirrakoi Rhino3D Medical https://mirrakoi.com/rhino3dmedical_for_engineers/ None

NemoFAB Nemotec www.nemotec.com None

OsiriX Lite Pixmeo www.osirix-viewer.com None

OsiriX MD Pixmeo www.osirix-viewer.com 3D

Visualization

Ossa 3D Conceptualiz http://www.conceptualiz.org/products_ossa.html None

Ricoh 3D for

Healthcare

Ricoh, USA, Inc https://www.ricoh-usa.com/en/industries/healthcare/3d-printing-

for-healthcare

3D Printing

Seg3D/

Biomesh3D

University of

Utah

https://www.sci.utah.edu/cibc-software/seg3d.html None

Simpleware Synopsis www.synopsis.com 3D Printing

Vitrea Vital Images/

Canon

www.vitalimages.com 3D

Visualization

3D printed models play a crucial role in

transforming surgical interventions by facilitating

pre-surgical planning and simulation,

intraoperative navigation, and physician training.

3D models are valued by many surgeons as they

provide spatial comprehension, tactile feedback,

and a better understanding of patient-speciﬁc

anatomical characteristics and variations

(Tzavellas et al. 2020). In conjunction with

computer-aided surgery simulation, 3D models

Description of method

are an invaluable tool in the preparation for a safer

and more cost-effective surgical pelvic recon-

struction minimizing operative time and

maximizing precision outcomes (Hung et al.

2019). Models manufactured with materials ful-

ﬁlling biocompatibility standards can be sterilized

using predeﬁned protocols and brought to the

surgical ﬁeld (Fang et al. 2019). Using 3D models

can allow for a reduction in overall operative and

ﬂuoroscopic times, a decrease in intraoperative

blood loss, increased accuracy of reduction and

ﬁxation and a decrease in complications such as

iatrogenic nerve injuries (Tzavellas et al. 2020).

Current 3D printing technologies can be limited

regarding material type and color capabilities,

although technologies are continuously improv-

ing, and it is expected that new material types and

color options will be more easily accessible and

affordable in the future.

6 Advanced 3D Visualization and 3D Printing in Radiology 109

Table 6.2 Description of 3D printing technologies

Printing technology

type

Binder Jetting Process in which a liquid bonding agent is selectively deposited to join powder materials.

Direct Energy

Deposition

Process in which focused thermal energy is used to fuse materials by melting as they are being

deposited.

Material Extrusion Process in which material is selectively deposited through a nozzle.

Material Jetting Process in which droplets of photosensitive build material are selectively deposited and

solidiﬁed with ultraviolet light.

Powder Bed Fusion Process in which thermal energy selectively fuses regions of a powder bed.

Sheet Lamination Process in which sheets of material are bonded to form a part.

Vat

Photopolymerization

Process in which liquid photopolymer in a vat is selectively cured by light-activated

polymerization.

6.6 3D Visualization and Modeling

for Cardiovascular Applications

Planning cardiovascular surgery is a collaborative

effort that integrates imaging expertise of

radiologists and clinical expertise of cardiologists

and cardiac surgeons to determine the best thera-

peutic options while optimizing operative time.

Over the last 10 years, 3D image visualization has

been used extensively for the planning of cardio-

vascular procedures such as transcatheter aortic

valve replacement (TAVR), widely established as

the most common FDA-approved treatment for

patients with severe symptomatic aortic stenosis

who are at moderate (or greater) risk for surgical

valve replacement (Hosny et al. 2019). This

endovascular procedure employs a catheter-

based delivery system using femoral arterial

access to implant a balloon-expandable

bioprosthetic valve within a diseased aortic

valve. Although in widespread use, the complex

3D anatomy of the aortic root makes it difﬁcult to

predict how the prosthetic aortic valve will adapt

in the patient and achieving a personalized pros-

thetic valve ﬁt for every patient remains a surgical

challenge. Oversizing the prosthetic valve can

lead to annular rupture, and under-sizing can

result in an ineffective seal/paravalvular leak or

prosthetic valve embolization. Characterization

of aortic root anatomy and aortic annular

measurements are an important starting point for

determining appropriate valve size, but additional

patient-speciﬁc factors such as the degree of

annular eccentricity and the relative distribution

of calciﬁed deposits on valve leaﬂets and in the

LVOT all inﬂuence valve anchoring and seal

efﬁcacy (Hosny et al. 2019). Figure 6.2 shows

aortic images from a patient with signiﬁcant

plaque on the annulus.

3D printed heart and aortic models are used

preoperatively and intraoperatively in the man-

agement of complex congenital heart disease

(CHD), acquired cardiovascular anomalies, car-

diac tumors and in percutaneous cardiology

procedures such as TAVR (Levin et al. 2020;

Yoo et al. 2015,2017; Marro et al. 2016). Ana-

tomic models facilitate improved spatial acuity to

critical structures with reduction in operative time

including cardiopulmonary bypass times. Opera-

tive rehearsals with patient-speciﬁc 3D printed

models reveal potential pitfalls to the surgeon,

the preferred approach and optimal instrumenta-

tion, while providing a preview of anatomic

nuances and opportunity to improve technique

(Yoo et al. 2017; Hussein et al. 2020; Hermsen

et al. 2017). 3D printed models assist in educating

surgical trainees by simulating speciﬁc anatomy

and allowing various surgical approaches to be

practiced in a stress-reduced environment without

harming the patient. Soft tissues can be mapped

and reconstructed, effectively moving important

decisions from the time-constrained operating

room to the preoperative setting (Costello et al.

2015; Ballard et al. 2018). Cardiothoracic

surgeons ﬁnd the models suitable for practicing

closure of septal defects, application of bafﬂes

(surgically-created tunnels used to redirect blood

ﬂow) within the ventricles, reconstructing the

aortic arch, and the arterial switch procedure

(Yoo et al. 2015). In addition, patient-speciﬁc

3D models are valuable for enhancing engage-

ment with parents and improving communication

between cardiologists and parents resulting is a

positive impact in parents’and patients’psycho-

logical adjustment to living with congenital heart

disease (CHD) (Biglino et al. 2015).

110 S. Fidvi et al.

Fig. 6.2 TAVR images

showing the annulus in

3 planes: (a) en-face with

the aortic valve, (b,c) aortic

valve outﬂow tract shown

from two angles. (d)3D

visualization of aortic

outﬂow tract and proximal-

mid aorta, (e) en-face 3D

visualization of the aortic

valve. Notice the plaque in

bright white on all images.

Images courtesy of Nicole

Wake, PhD, GE Healthcare

CHD such as Ventricular Septal Defects

(VSD), Tetralogy of Fallot, and Double Outlet

Right Ventricle (DORV) cause signiﬁcant hemo-

dynamic and functional consequences

necessitating surgical repair. 3D-printed replicas

of the patient’s heart permit precise understanding

of complex cardiac anatomy and hands-on simu-

lation of surgical and interventional procedures.

6 Advanced 3D Visualization and 3D Printing in Radiology 111

As an example, DORV (Fig. 6.3) is a cardiac

anomaly in which both the aorta and the pulmo-

nary artery originate predominantly or entirely

from the right ventricle. In this situation, the left

ventricle (LV) has no direct outlet to either great

vessel and must eject blood into the right ventricle

(RV) through a VSD. Rarely, in the absence of a

VSD, the LV is very hypoplastic. The physiolog-

ical picture and the type of surgical correction

depend on the arrangement of the great vessels

and the anatomy of the VSD, both of which are

well depicted by imaging. The most common

variant, the Fallot type, has a normal arrangement

of the great vessels, a subaortic VSD and is often

associated with pulmonic stenosis. DORV may

also be associated with an anterior aorta and a

subpulmonic VSD, called the Taussig-Bing

anomaly. Surgical correction for the Fallot type

variant consists of VSD patch closure which

directs blood to the aorta and correction of pul-

monic stenosis. For the Taussig-Bing variant

without pulmonary obstruction, surgical correc-

tion consists of patch closure of the VSD and

arterial switch. In the presence of pulmonary

obstruction, LV ﬂow is tunneled through the

VSD to the aorta and an RV-PA pathway is

created- the so-called Rastelli procedure (Quail

and Taylor 2020).

Fig. 6.3 Patient-speciﬁc 3D printed hollow cardiac model

printed with a ﬂexible material (TangoPlus, Stratasys) and

created from 3D cardiothoracic CT data obtained preoper-

atively in an infant with DORV and interrupted aortic arch

type B. Both the smaller AA and dilated MPA arise from

DORV. The aortic arch is interrupted (asterisk) between

the left common carotid artery and the left subclavian

artery, indicating type B of interrupted aortic arch. MPA

main pulmonary artery. Reproduced with open-access per-

mission from Goo et al. (2020)

3D-printed heart models are of value in train-

ing for and performing corrective surgery for

Hypertrophic Cardiomyopathy (HCM). HCM is

a genetic myocardial disease characterized by

marked hypertrophy of the LV and occasionally

the RV, possible obstruction of the LV outﬂow

tract (LVOT), life-threatening arrhythmias and

sudden cardiac death. Septal reduction therapies,

including surgical myectomy and percutaneous

transluminal septal myocardial ablation are

established treatments for drug refractory symp-

tomatic HCM. Poor visualization of the left ven-

tricular cavity in the operative ﬁeld and

heterogeneous LVOT anatomy can make this

surgical procedure challenging (Hamatani et al.

2017). The preferred method of treatment, surgi-

cal septal myectomy (SM), has potential

complications of iatrogenic ventricular septal

defect, residual left ventricular outﬂow tract

(LVOT) obstruction and mitral regurgitation

(MR) (Andrushchuk et al. 2018). Anatomic

models help avoid these complications by

optimizing visualization of complex intracardiac

morphology and the mitral apparatus and permit-

ting superior intraventricular septum resection

volume and shape (Ali et al. 2020). 3D-printed

HCM heart models also allow for patient-speciﬁc

preoperative simulation of a low-volume,

low-visibility, high-risk surgical procedure that

is traditionally difﬁcult to teach (Hermsen et al.

2017).

3D modeling is also commonly used in cardi-

ology for left atrial appendage (LAA) occlusion

(Goitein et al. 2017; Liu et al. 2016; Hong et al.

2022; Kim et al. 2022; Hachulla et al. 2019;

Morcos et al. 2018). The LAA is a common site

for thrombus formation in patients with atrial

ﬁbrillation (AF), the most common cardiac

arrhythmia. Its incidence increases with age to

>8% in those >80 years of age, and embolic

strokes originating in the LAA are associated

with a higher morbidity and mortality,

emphasizing the need for effective stroke preven-

tion in AF. While the mainstay of treatment is

pharmacological therapy with anticoagulants,

LAA occlusion/exclusion devices such as the

Watchman device (Boston Scientiﬁc,

Marlborough, MA) are a viable alternative in

patients in whom contraindications to anticoagu-

lant therapy exist or who are at high risk of

bleeding. Occlusion is deﬁned as placement of a

device into the LAA percutaneously, while exclu-

sion refers to the application of an external liga-

ture to isolate the LAA from the circulation

(Athanassopoulos 2016).

112 S. Fidvi et al.

The correct sizing and the optimal implanta-

tion position of the closure device pose a chal-

lenge due to complex and variable LAA shapes.

The risks of under- or oversizing are LAA perfo-

ration, pericardial effusion, interference with the

mitral valve or the ostium of the pulmonary veins,

compression of the left circumﬂex artery and

device embolization (Hachulla et al. 2019). Pre-

cise understanding and measurements of the

dimensions of the LAA ostium, landing zone

and maximum length of the main anchoring lobe

are essential elements in occlusion procedures

and especially important in patients with variant

anatomy (Fig. 6.4).

Obtaining accurate LAA dimensions is also

crucial to minimize intraprocedural

improvisations and device changes, recapture

maneuvers, and prolonged procedure times

(Morcos et al. 2018). Periprocedural noninvasive

imaging modalities utilized to evaluate the LAA

include ﬂuoroscopy, 2D or 3D transesophageal

echocardiography (TEE), intracardiac echocardi-

ography (ICE) and cardiac CT. 3D printed LAA

models are valuable tools in the identiﬁcation of

the optimal device size and position eliminating

incomplete sealing and peri-device leaks. Added

beneﬁts include technique optimization with sim-

ulation of the LAA closure procedure and a

reduction in the device implantation learning

curve (Hachulla et al. 2019).

Advanced 3D image visualization and printing

have also been used for patients with mitral

annular calciﬁcation (MAC), a degenerative pro-

cess involving the mitral annulus which

progresses to involve the leaﬂets and subvalvular

apparatus causing valvular obstruction or regur-

gitation. In patients with severe MAC who are not

surgical candidates, transcatheter mitral valve

replacement (TMVR) is a feasible therapeutic

option but remains surgically challenging due to

the complexity of the mitral valve apparatus and

its geometric relationship with the left ventricular

anatomy. The calciﬁed mitral annulus can vary in

rigidity, size, and shape, all of which can affect

anchoring of the deployed transcatheter valves

and predispose to paravalvular leaks (PVL). The

proximity of the mitral apparatus to the left ven-

tricle can lead to LVOT obstruction during

TMVR, either by direct prosthetic valve protru-

sion or by displacing the anterior mitral valve

leaﬂet into the LVOT. Furthermore, the bulky

calciﬁed annulus can lead to underexpansion

and dislodgement of the balloon-expandable

valves. Given the anatomical challenges, meticu-

lous pre-procedural 3D planning is warranted

(Wang et al. 2016,2018). 3D printed cardiac

models are valuable adjuncts to assess prosthesis

sizing, anchoring, expansion, paravalvular gaps,

LVOT obstruction and extra-annular calcium

extension (El Sabbagh et al. 2018) (Fig. 6.5).

Simulation with 3D models helps trial the com-

plex navigational maneuvers needed to advance

catheters from the aorta to the left ventricle,

preprocedurally select the size of the deﬂectable

guiding catheter and to optimize catheter tip posi-

tioning between the two papillary muscles and

close to the mitral annulus (Valverde 2017).

Another major use of advanced 3D image

visualization in cardiac applications is for cardiac

tumors. To safely resect or debulk a cardiac

tumor, it is imperative to understand its intricate

interplay with critical local cardiac structures to

help devise a safe surgical strategy while

protecting the coronary circulation. 3D printed

cardiac tumor models such as that shown in

Fig. 6.6 provide vital structural insight into

tumor size and location and are used to identify

the spatial relationship between the tumors and

the coronary arteries in addition to gauging their

depth and inﬁltration (Riggs et al. 2018).

6 Advanced 3D Visualization and 3D Printing in Radiology 113

Fig. 6.4 Example of a

cauliﬂower-shaped LAA.

(a)Cardiac CT and (b) TEE

which both predicted a

25-mm Amulet device.

Two different Amulet

sizes were tested on the 2D

printed model: (c) a 25 mm

sized device and (d)a

22 mm sized device

(Reproduced with open-

access permission from

Hachulla et al. (2019)

DCT=25 Amplatzer

DTEE=25 Amplatzer

25 Amplatzer

22 Amplatzer

Fig. 6.5 3D modeling and surgical planning for TMVR

in a patient with severe MAC. (a) cross-section of the

mitral annulus with estimated area measurement shown.

(b) 3D model of the hollowed left ventricle and atrium

with aortic outﬂow (red), left atrium (purple), and calciﬁed

plaque (yellow). (c) en-face projection of mitral valve

plane with simulated valve dark yellow. (d) hollowed

left-sided heart view showing mitral calciﬁcation. (e)CT

showing 4 chamber view with simulated valve (yellow

box), (f) CT showing en-face mitral annulus view with

simulated valve (yellow circle). (g) CT showing

2-chamber view with aortic outﬂow tract and simulated

valve (yellow box). (h) 3D model of the hollowed left

ventricle and atrium with the simulated valve placed

(light yellow). Images courtesy of Nicole Wake, PhD,

GE Healthcare

114 S. Fidvi et al.

Fig. 6.6 Photographs of a

physical 3D printed heart

model (white) with a tumor

(transparent) showing the

left circumﬂex artery

coursing through the tumor

[Reproduced with open-

access permission from

Riggs et al. (2018)]

Finally, open surgical repair of abdominal aor-

tic aneurysms (AAA) has given way to catheter-

based endovascular aneurysm repair (EVAR)

which requires specialized skills and training

(Bastawrous et al. 2018). Endovascular repair of

complex aortic aneurysms involving the origin of

aortic branches, extreme angulations, complex

aneurysm neck anatomy, and short landing

zones can be technically challenging. Pertinent

information sought from preoperative CT

angiograms includes assessment of access (com-

mon femoral artery) for its diameter (large

enough to accommodate delivery system), pres-

ence of plaques or calciﬁcations which can hinder

access, iliac artery diameters at landing zones and

iliac vessel tortuosity (Chepelev et al. 2015).

Simulation on 3D printed aortic phantoms is ben-

eﬁcial in the preoperative planning and teaching

of aneurysm repairs by identifying the best

projections for angiography, the best catheter

and wire combinations to navigate the anatomy

and in determining appropriate stent size, design,

and position (Sommer et al. 2018,2021; Meess

et al. 2017) (Fig. 6.7). Haptic feedback elicited

from manipulation of guidewires and catheters

also aids complex endovascular interventions,

increasing operator conﬁdence (Chepelev et al.

2018).

6.7 3D Visualization and Modeling

for Musculoskeletal

Applications

3D image post-processing is routinely performed

to better visualize musculoskeletal pathologies

such as complex shoulder fractures (Fig. 6.8).

3D printed-assisted operations for comminuted

and intraarticular proximal humeral fractures

shortened surgery duration and time to fracture

union, improved healing in an anatomical posi-

tion and shoulder function, reduced blood loss

volume, the number of ﬂuoroscopies during sur-

gery and minimized postoperative complications

(Li et al. 2022).

In the setting of pelvic trauma, 3D visualiza-

tion of the fractured hemi-pelvis provides

surgeons an accurate understanding of fracture

conﬁguration, allowing the manipulation of

bone fragments in a virtual simulation and crea-

tion of personalized treatment plans utilizing the

best surgical approach and reduction technique as

well as optimal interfragmentary screw

trajectories (Hurson et al. 2007). Surgery around

the bony pelvis poses unique challenges due to

complex anatomy, deep exposures and narrow

safe corridors required to avoid critical

neurovascular and visceral structures (Fang et al.

2019); and 3D visualization is a much more pow-

erful tool as compared to traditional radiological

assessment of pelvic fractures involving plain

X-rays and CT (Hurson et al. 2007).

6 Advanced 3D Visualization and 3D Printing in Radiology 115

Fig. 6.7 Case with an abdominal aortic aneurysm with a

dissection showing image contours generated from image

segmentation and subsequent hollowing of the blood pool

on the (a) coronal, (b) axial, and (c) sagittal CT images. (d)

Virtual 3D model of the anatomic region of interest. (e)3D

printed model of the printed aortic aneurysm model

printed with jet fusion technology. Images courtesy of

Nicole Wake, PhD, GE Healthcare

Fig. 6.8 Humeral head fracture (yellow arrow) shown on (a) axial, (b) coronal, and (c) sagittal CT images with (d)

corresponding 3D model of the fracture. Images courtesy of Nicole Wake, PhD, GE Healthcare

116 S. Fidvi et al.

3D printing the mirror image of the opposite

intact anatomy can also play an invaluable role in

the accurate pre-contouring of ﬁxation plates,

ensuring optimal implant positioning, minimizing

dissection of adjacent soft tissues, reducing sur-

geon fatigue, and minimizing the need for implant

repositioning (Fang et al. 2019; Hung et al. 2019).

An example of mirroring is shown in Fig. 6.9.

3D visualization is also commonly used to

reconstruct the bony anatomy for joint replace-

ment and repair, such as the knee and shoulder.

Using CT data, these 3D reconstructions allow

improved comprehension of conditions such as

osteoarthritis and allow for precise surgical

planning using custom devices that match the

patient’s speciﬁc anatomy (Berhouet et al. 2017;

Koch et al. 2021; Kwon et al. 2005; Lei et al.

2020). In patients with shoulder osteoarthritis,

disproportionate posterior load bearing deforms

the glenoid leading to bony thinning, retrover-

sion, and biconcavity of its articular surface. Sur-

gical techniques for shoulder replacement surgery

total shoulder arthroplasty (TSA), shoulder

hemiarthroplasty (SHA), and reverse shoulder

arthroplasty (RSA) with adjunctive measures

such as bone grafting and glenoid reaming. Sur-

gical objectives include restoration of normal

glenoid version and osteophyte removal to rebal-

ance the humeral head, restore normal biome-

chanics, achieve durable hardware ﬁxation, and

provide lasting symptomatic relief and functional

ability. The small volume of glenoid bone stock

in conjunction with complex biomechanical

forces and its relative avascularity and low

strength (Wang et al. 2019) leads to loosening

of the glenoid component, which is widely

recognized as the primary limitation in the mid-

and long-term durability of TSA (Al Najjar et al.

2018). The bony morphology of the glenoid is

therefore a critical factor in the selection of appro-

priate surgical technique and preoperative

planning for implant placement to ensure

arthroplasty durability. As shown in Fig. 6.10,

3D printed models create realistic anatomic-

scale representations of glenoid morphology

including osteophytes, bony thinning, retrover-

sion, and biconcavity.

3D visualization and modeling are also used in

patients with spinal deformities such as idiopathic

scoliosis, kyphosis, and meningomyeloceles to

facilitate the study of joint inclination, false

articulations and pedicle size (Fig. 6.11). In the

preoperative setting, these models serve as a

guide to curve correction and pedicle screw place-

ment, resulting in a safer and more optimized

surgical outcome.

Patient-speciﬁc 3D printed models can be

highly valuable in cases of orthopedic oncology

(Fig. 6.12). In addition, in these cases, surgical

guides can also be created directly from volumet-

ric images to guide corrective bone resections or

complex tumor resections. In addition, as recon-

structive and restorative surgeries have advanced

in scope and complexity in the ﬁeld of Orthope-

dics, 3D printing technology holds the potential

for bringing personalized medicine and patient-

centered healthcare closer to reality by enabling

the fabrication of patient-speciﬁc implants.

Despite the availability of multiple standardized

commercial implants, a subset of patients fall

outside the window of available devices and

beneﬁt from the design and production of individ-

ually tailored prostheses and orthotics (Mitsouras

et al. 2015). Consequences of an improperly ﬁtted

implant include patient embarrassment and dis-

comfort, decreased patient compliance and even

depression (Ballard et al. 2018).

At this time, most implants created using addi-

tive technologies are created by original equip-

ment manufacturers with 510 K FDA clearance to

manufacture and sell the intended part. Typically,

the design of a patient-speciﬁc implant utilizes the

concept of “Mirroring”to create a template of

patient anatomy in the setting of severe patho-

logic unilateral deformities. As humans exhibit

plane symmetry though the midline sagittal

plane, the non-pathologic side can be mirrored

and computationally overlapped with the dis-

eased side allowing rapid and accurate patient-

speciﬁc reconstruction. This technique enables

the creation of patient-speciﬁc surgical guides

which allow the precise excision of bone and

soft tissue tumors, optimization of the resection

volume, conservation of healthy tissue and

subsequent placement of the custom implant

ﬂush with the excision site (Chepelev et al. 2015).

3D-printed implants are used for tumor

endoprosthetic reconstruction in cases of

osteosarcomas, pelvic chondrosarcomas and

tumors of the spine, clavicle, scapula and calca-

neus. Custom-made implants are accompanied

with a set of individualized tools for replicating

the planned bone cuts, custom-made trial

implants and drill guides to facilitate accurate

placement of the prosthesis. Full-scale models

for preoperative planning and intraoperative ref-

erence and guidance are also provided. Accurate

intraoperative placement can, however, be chal-

lenging due to absence of intraoperative ﬂexibil-

ity and the design and manufacture of these

models are time-intensive and costly (Fang et al.

2019).

6 Advanced 3D Visualization and 3D Printing in Radiology 117

Fig. 6.9 (a) Axial CT image demonstrating tumor (yel-

low arrow) with corresponding 3D model with bone (yel-

low) and tumor (blue). (b) Image of the left side, mirrored

to the right, with the plane vertically at the center of the

nose. (c) Mirror image depicted in purple with the original

bone (yellow) and tumor (blue). Note that rotation would

be performed next to ensure proper alignment. Images

courtesy of Nicole Wake, PhD, GE Healthcare

Finally, 3D printing of personalized orthotics

(externally worn medical devices used to modify

the structural and functional characteristics of the

neuromuscular and skeletal system) using medi-

cal imaging data can allow for personalized

solutions that can be advantageous as compared

to standard devices. Orthotics are typically used

in the setting of cerebral palsy, stroke, traumatic

brain injuries, multiple sclerosis, and clubfoot to

support the lower leg and correct imbalance. 3D

printing technology offers design freedom and

manufacture of patient-speciﬁc orthotics with the-

oretically optimized biomechanics, improved

functionality, better ﬁt, and aesthetics. Their rela-

tively low cost is an important factor for children

requiring multiple replacements as they grow

(Matsumoto et al. 2015).

6.8 3D Visualization and Modeling

for Abdominal Applications

The use of 3D printing in abdominal interventions

and clinical scenarios is becoming increasingly

common, with applications for surgical planning

at the forefront. Image segmentation is challeng-

ing in abdominal structures as they demonstrate

similar grayscale shades, which renders differen-

tiation between them and their surrounding

vessels and fat difﬁcult to process. Many solid

organ tumors have similar grayscale appearances

compared to the surrounding organ parenchyma,

which makes delineation difﬁcult. In addition,

hepatic parenchyma, vascular, and biliary

structures are closely opposed to each other and

can often overlap or are difﬁcult to distinguish on

conventional 2D images. Furthermore, the vascu-

lar and biliary structures often take oblique,

non-linear courses which are similarly challeng-

ing to comprehend on 2D images (Mitsouras et al.

2015). However, medical teams ﬁnd 3D

modeling beneﬁcial as abdominal anatomy can

be relatively complex.

118 S. Fidvi et al.

Fig. 6.10 3D model printed of the scapula created with

selective laser sintering printing technology. (a) View

from lateral demonstrates a large anteroinferior glenoid

osteophyte (arrow). (b) View from inferior shows glenoid

retroversion and posterior bony thinning (arrows). (c)3D

model printed using a stereolithography printer. View

from inferolateral demonstrates glenoid biconcavity with

a ridge between the two depressions in the glenoid surface

(dotted line). Reproduced with permission from Wang

et al. (2019)

For abdominal applications, the two organ

systems for which 3D modeling is most widely

performed are hepatobiliary and renal structures

(Mitsouras et al. 2015). These organs are clearly

visible in medical images (CT and MRI) with

smooth, regular borders that make them amenable

to segmentation and reconstruction (Pietrabissa

et al. 2020).

Liver transplant surgeries have been a frequent

subject of 3D modeling (Mitsouras et al. 2015).

There is a relative paucity of cadaveric donors for

the transplant procedures, and many transplant

procurement operations rely on living, healthy

individuals undergoing lobectomies, which can

have high morbidity. Much of this morbidity is

due to inaccurate preoperative characterization of

biliary and vascular structures and suboptimal

estimates of liver volume (Zein et al. 2013).

6 Advanced 3D Visualization and 3D Printing in Radiology 119

Fig. 6.11 (a) Shaded surface volume rendering of a

patient with severe scoliosis. (b) Segmentation and visual-

ization of a patient with scoliosis and a butterﬂy vertebra.

right). Images courtesy ofNicole Wake, PhD, GE

Healthcare

Determining the volume of the donor’s liver

and individual lobes is an important component

of operative planning as it ensures adequate post-

operative reserve. Traditionally, these volumes

are generated using modeling software on com-

puter workstations (Fig. 6.13), although several

studies have shown that 3D printed models have

been more accurate in assessing liver volumes

when compared to ex-situ surgical specimens

(Zein et al. 2013). The same volumetric calcula-

tion and anatomic delineation can help when

large portions of liver must be resected for other

reasons than transplantations, such as solitary

tumor resections. The tumor’s relationship to

adjacent vascular and biliary structures, as well

as location within a segment, can be clariﬁed with

these printed models (Fang et al. 2015).

3D modeling also aids in the planning of par-

tial nephrectomies for renal tumor resections

(Silberstein et al. 2014). The kidney normally

has an oblique position within the upper abdo-

men, which can be difﬁcult to visualize on 2D

imaging, and has multiple vascular and excretory

structures that must be carefully considered in

surgical planning, similar to the liver. 3D printing

clariﬁes the relations between tumors and these

structures for preoperative planning (Fig. 6.14)

(Mitsouras et al. 2015; Wake et al. 2017,2018,

2019). 3D printed models have also been used to

plan the removal of kidneys from living donors in

renal transplantation planning, as well as the

removal of renal stones percutaneously

(Pietrabissa et al. 2020).

The same goals of elucidating tumor location

and relationship to adjacent structures have been

described in the resection of pancreas, spleen, and

bowel tumors and in treating anorectal ﬁstulae

(Marconi et al. 2017; Hamabe and Ito 2017).

The technology has also been implemented in

preoperative planning of abdominal wall hernias

and surgical mesh development (Ballard et al.

2018).

120 S. Fidvi et al.

Fig. 6.12 Patient with a complex pelvic tumor showing

(a) axial view with bone segmentation (red) and tumor

segmentation (green), (b) 3D visualization of the left

hemi-pelvis with tumor in the posterior and anterior

views, (c) 3D visualization of the whole pelvis with

tumor, and (d) Posterior and anterior photographs of the

3D printed left hemi-pelvis model printed with material

extrusion technology with the bone (white) and tumor

(pink). Images courtesy of Nicole Wake, PhD, GE

Healthcare

3D imaging is also commonly used during

virtual colonoscopy studies for colon cancer

screening. During these exams, carbon dioxide

gas or room air is insufﬂated directly into the

colon and a CT scan is performed in the axial

plane. These conventional 2D axial images are

then often processed to generate 3D views to

enhance polyp detection (Geenen et al. 2004).

These views include endoluminal displays,

which mimic the viewpoint of conventional endo-

scopic colonoscopy, or virtual dissection views

which display the entire lumen surface as a ﬂat-

tened image (Silva et al. 2006) (Fig. 6.15).

For prostate cancer, MRI is often used to local-

ize lesions for biopsy, and many clinicians

attempt to determine whether the cells detected

in the resected specimen after prostatectomy

match the ﬁndings predicted on the preoperative

MRI. The difﬁculty in comparing the resected

gland to the MRI is that the prostate is very soft

and often deforms when removed, rendering its

shape and morphology different than that on

imaging. To ameliorate this pitfall, institutions

have developed MRI-based patient-speciﬁc3

models to serve as a mount for the removed

gland (Makinen and Makinen 1971; Newman

1971; Wu et al. 2019). The model holds the

prostate in place as it is sliced to match the loca-

tion and orientation of the MRI lesions. One study

found that using these patient-speciﬁc mounts

was more accurate in correlating histology and

MRI imaging than using standard slicing

techniques (Costa et al. 2017). Some urologists

also use 3D models for preoperative planning of

prostatectomies and partial gland treatment, par-

ticularly in assessing the location of

6 Advanced 3D Visualization and 3D Printing in Radiology 121

Fig. 6.13 Automated liver volume analysis performed on the GE AW workstation showing liver segmentation, 3D

visualization, and total volume calculation. Images courtesy of Nicole Wake, PhD, GE Healthcare

Fig. 6.14 Patient with Tuberous Sclerosis and multiple

angiomyolipomas showing (a) coronal MR image with a

left upper pole mass (Tuberous Sclerosis complex

associated renal cell carcinoma) highlighted in green, (b)

3D model shown on computer screen with a lattice design

for the kidney parenchyma, and (c) photograph of the 3D

printed model. The kidney parenchyma was designed as a

lattice structure so the internal anatomy could be properly

visualized in the 3D printed model which was printed with

binder jetting technology (CJP 660, 3D Systems). Images

courtesy of Nicole Wake, PhD, GE Healthcare

neurovascular bundles and of the bladder neck to

ensure that these are avoided (Jomoto et al. 2018;

Wake et al. 2016,2020,2021).

122 S. Fidvi et al.

Fig. 6.15 CT Colonography with 3D processing. (a)

supine and (b) prone axial CT images demonstrating a

polyp in the ascending colon (arrow). (c)3

reconstruction highlights the polyp (arrow). Images cour-

tesy of Dr. Judy Yee, Monteﬁore Medical Center

6.9 3D Visualization

for Neurological Applications

Neurological disorders such as brain tumors,

strokes, and hemorrhage represent a major global

health burden. In neuroradiology, 3D reformats of

CT head and neck images are commonly used

clinically for improved visualization of complex

facial fractures and vertebral body injuries, where

these reconstructions have been shown to

improve the delineation of fracture line extent

and displacement (Kaur and Chopra 2010). 3D

reconstructed fracture maps of thoracic and lum-

bar vertebral bodies also improve understanding

of fracture patterns to improve clinical decision-

making (Su et al. 2019).

Similar to conventional CT, CT angiography

(CTA) examinations are crucial for the evaluation

of cerebrovascular pathology. In cases of multiple

intracranial aneurysms, CTA source images can

be used to determine the pattern and location of

hemorrhage, as well as to determine the acuity of

hemorrhage and the presence of active bleeding

(Sanelli et al. 2005).

MRI is another valuable data source for high-

resolution visualization of neuroanatomy.

Techniques imaging the cerebral vasculature

such as MR angiography (MRA) or contrast-

enhanced MRA are now increasingly utilized in

their 3D form to help provide a more comprehen-

sive analysis prior to surgical and/or endovascular

interventions. For the surgical management of

meningiomas and intracranial arteriovenous

malformations, high temporal and spatial resolu-

tion 3D MRA studies have been shown to reduce

the number of invasive diagnostic angiograms by

identifying and classifying ﬁstulas, and by

illustrating major feeding arteries in the evalua-

tion of the meningiomas (Reinacher et al. 2007).

In addition, MR techniques such as diffusion

spectrum imaging can also be used to characterize

the 3D diffusional displacement of water

molecules, allowing for enhanced comprehension

of the connectivity pathways in the brain

(Wedeen et al. 2005; Callaghan 1993). Advanced

image visualization and analysis can also help to

quantify brain structures (e.g., tumor size and

shape) and highlight their subtle changes

over time.

6.10 3D Printing in Neurosurgery

Studies reporting on 3D printing in neurosurgery

have primarily been focused on the creation of

patient-speciﬁc models for surgical planning

(Fig. 6.16). Surgical planning for brain tumor

resection typically involves using MRI to sepa-

rate tumor and surrounding normal tissue. Despite

this technology, anatomic relationships

demonstrated on 2D MRI images may still be

difﬁcult to appreciate before and during the pro-

cedure. 3D printing technology permits MRI data

to be converted to patient-speciﬁc models,

improving comprehension of complex anatomic

relationships between tumor, blood vessels, bone,

and adjacent normal tissue, and facilitating

surgeons’conceptualization of the location and

extent of neoplastic tissue (Oishi et al. 2013;

Spottiswoode et al. 2013) (Fig. 6.17).

6 Advanced 3D Visualization and 3D Printing in Radiology 123

Fig. 6.16 Multi-colored 3D printed model of a skull base

tumor (blue) shown alongside associated vasculature (red)

and bone (clear). The model was printed using material

jetting and is shown with corresponding images from the

(a) side view and (b) top-down view. The model helped to

guide the surgical approach for tumor resection. Images

courtesy of Nicole Wake, PhD, GE Healthcare

Printed neurological models illustrating the

associations of tumor to its surrounding

anatomical structures have also allowed surgeons

to simulate surgical approaches (Oishi et al.

2013). For ventriculostomy procedures,

3D-printed simulators are a cost-effective alterna-

tive to virtual haptic simulators while providing a

realistic training experience (Bova et al. 2013;

Ryan et al. 2015; Waran et al. 2014c,2015).

These simulators involve a reusable base segment

and a disposable segment for practicing the tech-

nique. Some of these devices involve using a

ﬂuid-ﬁlled ventricular system providing variable

pressure to simulate pathology (Ryan et al. 2015;

Waran et al. 2015). Another simulator makes use

of an electromagnetic tracking system registered

to a virtual image of positioning once the skin,

skull and dura are traversed (Bova et al. 2013).

An additional role of 3D printing in simulation

development is in the ﬁeld of transsphenoidal

endoscopic pituitary removal. Multiple studies

have made use of skull replicas to practice this

surgical approach (Inoue et al. 2013; Waran et al.

2012). The printed skulls are registered to surgi-

cal navigation software to better depict the surgi-

cal procedure and to allow the model to be paired

in real-time with the neuroimages (Waran et al.

2012,2014c).

With the shift towards endovascular treatment

of aneurysms, simulation plays a key role for

trainees due to the lack of realistic cadaveric

tissue. 3D printed replicas of hollow elastic

aneurysms improve trainees’procedural expertise

by offering the opportunity to visualize aneurysm

shape and to practice aneurysm clipping

(Mashiko et al. 2015). A more realistic simulation

of the different tissue types is provided by using

materials with varying densities and

consistencies. Li et al. found in a large-scale

study that medical students using 3D printed

models had a signiﬁcantly improved understand-

ing of fracture anatomy when compared with

students using conventional CT images, but no

difference in the use of 3D models vs. virtual

renderings (Mashiko et al. 2015). Finally, 3D

printed neurosurgical models have also been use-

ful for training for brain biopsies, with studies

demonstrating that with the use of 3D printed

simulators, less experienced trainees required

shorter duration and fewer attempts to complete

the task (Waran et al. 2014a,b).

124 S. Fidvi et al.

Fig. 6.17 3D printed

cervical spine tumor

(C2 chordoma) model

showing (a) axial MR

image with tumor

segmentation (green), (b)

axial CT image showing

bone and vertebral artery

segmentation (yellow and

red respectively), (c)

Virtual 3D model showing

the bone (white), vertebral

arteries (red) and tumor

(green), and (d) 3D printed

model printed in multiple

colors with binder jetting

technology (CJP 660, 3D

Systems, Rock Hill, SC).

Images courtesy of Nicole

Wake, PhD, GE Healthcare

6.11 Digital Technologies

in Craniomaxillofacial Surgery

Virtual oral and maxillofacial surgeries using

templating with 3D technologies originated in

the mid-1990s (Xia et al. 2000). Digital

osteotomies are simulated in computer-aided-

design software during these procedures and

transferred directly to the patient with a patient-

speciﬁc surgical guide designed from the medical

images. Providing personalized, tailored

solutions in the operating room can improve sur-

gical accuracy and minimize surgical time,

thereby improving patient outcomes (Ballard

et al. 2020, Arce et al. 2020, Alexander and

Wake 2022). Common surgical procedures that

utilize these technologies include orthognathic

surgery (also known as corrective jaw surgery),

distraction osteogenesis (a treatment to lengthen

the craniofacial skeleton), and free ﬂap recon-

struction (Christensen 2018).

In orthognathic surgery, 3D printing has been

used to create anatomic models, occlusal splints,

osteotomy guides, repositioning guides, ﬁxation

plates/implants, and spacers (Lin et al. 2018). For

distraction osteogenesis procedures, 3D-printed

surgical guides decrease operative and ventilation

times, as well as reduce hospital stays compared

to traditional treatments (Mao et al. 2019). In

mandibular free ﬂap procedures, the jaw is rebuilt

using either autologous or donor bone. The most

common autologous bone used for this purpose is

the ﬁbula, the smaller of the two bones in the

lower leg. Creating patient-speciﬁc cutting guides

from CT data precisely delineates the length and

angles at which both the mandible and ﬁbula are

cut and optimizes autologous bone ﬁtting to the

resected area (Alexander and Wake 2022).

6 Advanced 3D Visualization and 3D Printing in Radiology 125

6.12 Intraoperative Navigation

Intraoperative navigation is a valuable tool for

complex neurosurgical procedures such as cranio-

facial surgeries, orthognathic procedures and

endoscopic sinus surgeries to ensure proper

tumor resection, bone segment repositioning,

and timely bone graft reconstruction (Andrews

et al. 2015; Zavattero et al. 2015; Bolzoni Villaret

et al. 2014).

CT or MRI-guided stereotactic neurosurgery

enhances safe and accurate targeting of deep brain

structures. Neurosurgeons can insert a probe into

many lesions or anatomic locations in the brain

through this imaging guidance to perform

location-speciﬁc procedures such as lesion

biopsy, abscess drainage, pharmaceutical agent

instillation, or electrode implantation (Lunsford

2012). In otolaryngology, 3D stereotactic guided

sinus surgery is routinely used in the ﬁeld of

endoscopic sinus surgery. Due to the fatality of

the complications which can arise from endo-

scopic sinus surgery, even the most experienced

surgeons value as much information as possible

intraoperatively. Clinically, studies have found

that the various CT/MRI-guided navigation

systems have an accuracy of approximately

1 mm and have the beneﬁt of increased margin

of intraoperative safety (Caversaccio and

Freysinger 2003).

6.13 3D Visualization and Modeling

for Breast Applications

Breast cancer is impacted by a multitude of

factors, including cancer stage, prognostic factors

and patient-speciﬁc factors (Santiago et al. 2019).

Early-stage breast cancer is typically treated with

breast conservation surgery (BCS) using a com-

bination of radiation and/or chemotherapy in

place of a mastectomy. Treatment is guided by

the need to balance achievement of tumor-free

margins with satisfactory cosmesis (Santiago

et al. 2019). The utilization of 3D imaging for

breast cancer management, including CT, MRI,

and 3D mammography, allows for optimized

patient autonomy, improved tumor-free margins

for BCS, and advances in treatment. Furthermore,

the ability for a patient to hold a tangible depic-

tion of their breast lesion can help patients under-

stand the potential treatment options for their

cancer and allow them to take an active role in

decision-making (Fig. 6.18).

3D imaging and printing provides the surgeon

with a more complete conceptualization of a

breast lesion, reduces operating times, and limits

the number of repeat operations (Schulz-

Wendtland et al. 2017) Utilizing MRI informa-

tion, a study created personalized 3D printed

models and bra-like plastic locators, allowing

surgeons to mark the edges of a tumor on the

breast surface and to inject blue dye into the

breast 1 cm from the tumor edges (Barth Jr. et al.

2017). This technique allowed for accurate local-

ization of 18 of 19 cancers with all 68 blue dye

injections sufﬁciently beyond the tumor edges

(Barth Jr. et al. 2017). An additional study

demonstrated that the utilization of 3D printed

molds as a guide for BCS resulted in preserving

normal breast tissue while achieving negative

margins (Rao et al. 2018). Further, a study has

shown potential for semi-automated delineation

of a breast lesion using MRI information to create

subsequent 3D printing (Schulz-Wendtland et al.

2017), which has potential to expedite

prototyping.

The literature provides a case of breast cancer

that was favored to be treated with mastectomy,

as BCS was thought to necessitate signiﬁcant

breast size alteration due to the extent of disease

(Santiago et al. 2019). Upon review of a 3D

printed model of this particular patient’s tumor,

the patient and surgeon agreed on BCS, and not

only was adequate cosmesis achieved, but post-

operative imaging demonstrated no recurrent dis-

ease at 6 months (Santiago et al. 2019). This

example illustrates the utility of 3D printing for

complex breast cancer patients, and explains why

institutions, such as MD Anderson routinely cre-

ate models, as seen in the provided examples

(Figs. 6.19,6.20, and 6.21).

Accurate surgical resection relies on palpation,

which can make for imprecise breast lesion local-

ization (Schulz-Wendtland et al. 2017). Though

preoperative lesion size assessments are accurate

for most women, numerous factors including

tumor size and breast density may inﬂuence pre-

operative assessment of a breast lesion (Schulz-

Wendtland et al. 2017). Often a woman will have

a wire localization procedure prior to excision of

nonpalpable breast cancer, wherein a radiologist

localizes the cancer’s location using a wire with

imaging guidance. Alternatively, localization can

be performed with alternate markers, such as

radioactive seeds and radiofrequency identiﬁca-

tion tags. Despite the utilization of wire localiza-

tion, the technique still requires the surgeon to

estimate the 3D position of a cancer from 2D

mammographic images (Barth Jr. et al. 2017).

Furthermore, wires may enter the breast at a

substantial distance from the site of the cancer.

It follows that this imprecision results in positive

margins requiring re-excisions in 22 to 34% of

cases (Barth Jr. et al. 2017; Lovrics et al. 2011;

Schnabel et al. 2014; Chagpar et al. 2015; Rao

et al. 2018), and in some subgroups, the repeat

excision rate can be as high as 40% (Schulz-

Wendtland et al. 2017).

126 S. Fidvi et al.

Fig. 6.18 Patient with a

large breast mass showing

(a) 3D visualization of the

skin (pink) shown with the

segmented tumor (purple),

and arterial supply (red). (b)

3D printed model of the

tumor and vasculature. (c)

3D printed model of the

tumor (red) shown with the

breast from the chest wall

perspective. Images

courtesy of Nicole Wake,

PhD, GE Healthcare

3D imaging also has potential treatment

advances for breast cancer. High dose rate

(HDR) breast brachytherapy, radiation utilized

for breast cancer treatment, can potentially be

optimized utilizing 3D technology. For instance,

a study found that using a combination of a cath-

eter optimization procedure and a computer-

controlled robotic 3D ultrasound system allowed

for real-time, rapid guidance and planning for

treatments down to 14 and 12 catheters (Poulin

et al. 2015). Additionally, utilization of a 3D

template has been shown to be a safe and repro-

ducible method to assess the lesion volume for

HDR multicathether brachytherapy (Aristei et al.

2019). Finally, 3D printing has potential to reduce

heart and lung radiation doses during post-

mastectomy radiotherapy, via creating

customized virtual boluses of electron beam ther-

apy (Yang et al. 2019). These results are impor-

tant as the rate of ischemic heart disease has been

shown to be proportional to the mean cardiac

dose of radiotherapy for breast cancer (Darby

et al. 2013), thus dose optimization can have a

potentially profound impact on long-term longev-

ity of breast cancer survivors.

6 Advanced 3D Visualization and 3D Printing in Radiology 127

Fig. 6.19 68-year-old woman with left breast invasive

ductal carcinoma with micropapillary and mucinous

features and DCIS cribriform with micropapillary growth

patterns and comedonecrosis. (a) Lateromedial mammo-

gram demonstrates a focal asymmetry (rectangle). (b)

Sagittal dynamic contrast-enhanced MRI demonstrates

non-mass clumped enhancement (rectangle) in the upper

inner quadrant with a post-biopsy clip corresponding to

the focal asymmetry noted by mammography. (c) Lateral

views of the 3D printed model with (left) overlying skin

obscuring the tumor detail and without (right) the

overlying skin, both printed at 65% scale with the skin

(clear), tumor (yellow), and pectoris muscle (gray). Sup-

port structures for the tumor arise from the medial skin

(asterisk). (d) Photograph of the patient with the models

demonstrating signiﬁcant lateral gland displacement when

the patient is in the supine position. Disease localized

wires (numbered) corresponding to the area of disease

(yellow) in the 3D printed model. Images courtesy of

Dr. Lumarie Santiago, MD Anderson Cancer Center

6.14 3D Visualization and Modeling

for Fetal Medicine

While 2D Ultrasound is of paramount importance

in the examination of fetal anatomy and antenatal

detection of congenital anomalies, 3D ultrasound

and MRI are important complementary imaging

modalities which can enhance pregnancy man-

agement in select congenital fetal malformations.

The two main barriers to high-quality obstetri-

cal 3D ultrasound are bone shadowing and fetal

128 S. Fidvi et al.

Fig. 6.20 72-year-old woman with the left breast DCIS

solid type with focal cribriform differentiation with focal

central necrosis and involvement by calciﬁcations preop-

erative imaging including mammogram and breast MRI.

(a) Lateromedial mammogram demonstrates a focal asym-

metry associated with calciﬁcations in the left breast

(oval). (b) Dynamic contrast-enhanced sagittal breast

MRI image of the left breast demonstrates non-mass,

clumped enhancement in the upper outer quadrant (oval)

corresponding to the focal asymmetry and calciﬁcations

noted by mammography. (c) lateral view of the 3D printed

model depicting I-125 seed placement in red

corresponding to their location in the post-procedure

mammogram circles. Images courtesy of Dr. Lumarie

Santiago, MD Anderson Cancer Center

Fig. 6.21 Photographs of the patient from this ﬁgure

showing (a) Signiﬁcant lateral displacement of the breast

with I-125 seed activity marked on the skin arrows. (b)

Circumvertical incision provided excellent exposure for

excision of the bracketed area of disease. (c) Smaller

scale 3D printed model obtained from imaging and prone

position versus expected ptosis from macromastia in

supine position. Images courtesy of Dr. Lumarie Santiago,

MD Anderson Cancer Center

movement, both of which require a high degree of

operator skill. The lack of uniformity in industry

standards regarding the storage format for volume

datasets generated by 3D ultrasound also limits

widespread use (Gonçalves 2016).

6 Advanced 3D Visualization and 3D Printing in Radiology 129

3D ultrasound allows superior depiction of

fetal surface anomalies and contributes to the

improved ﬁrst-trimester prenatal diagnosis of

neurological defects (anencephaly, acrania,

encephalocele, and holoprosencephaly), craniofa-

cial anomalies (orofacial clefts and cyclopia),

abdominal wall defects (omphalocele and

gastroschisis) and musculoskeletal dysplasias.

The more severe malformations are an important

cause of childhood mortality and morbidity, with

the latter having long-term ﬁnancial and psycho-

social implications for the affected child and their

family. The use of 3D ultrasound, particularly the

surface rendering mode, allows parents to better

conceptualize the malformation, thereby enhanc-

ing their ability to participate in prenatal

counseling and make informed decisions (Araujo

Júnior et al. 2015).

Figure 6.22 illustrates this concept with an

example of anencephaly, a lethal open neural

tube defect characterized by absence of the cra-

nial vault with variable amounts of angiomatous

stroma at the base of the skull (Callen 2009;

Nyberg et al. 2003; Akinmoladun et al. 2020).

Holoprosencephaly is a rare congenital brain

malformation characterized by partial or total fail-

ure of separation of the brain tissue in early

development. In its most severe form, alobar

holoprosencephaly, cleavage fails with a resultant

single midline forebrain with a primitive

monoventricle, often associated with a large

dorsal cyst (Winter et al. 2015). Associated cra-

niofacial anomalies include proboscis (a snout-

like protrusion from the face/forehead) and

cyclopia (a single palpebral ﬁssure and a single

midline orbit) as depicted in Fig. 6.23 (Sepulveda

et al. 2012).

Visualization of the fetal face is an important

component of obstetrical 2D ultrasound which

can diagnose and classify orofacial clefts (Callen

2009, Nyberg et al. 2003) by depicting the fetal

proﬁle, upper lip and alveolar ridge in the sagittal,

coronal, and axial views, respectively. The advent

of 3D ultrasound with its surface rendering mode

(Fig. 6.24) can generate detailed views of the fetal

face at different stages of development, allowing

the diagnosis of cleft lip and palate as early as the

ﬁrst trimester when a ﬂat palate and absence of

shadowing from adjacent non-ossiﬁed bones

permits better visualization (Sepulveda et al.

2012; Werner et al. 2010).

The excellent soft tissue contrast capabilities

of MRI have led to it surpassing ultrasound in the

diagnosis and prognostication of congenital fetal

anomalies of the brain and heart. The principal

disadvantages of MRI are image data disruption

due to fetal motion and maternal breathing. To

minimize these, fetal MRI acquisition protocols

are restricted to two-dimensional sequences with

high-resolution reconstruction of 3D motion-

corrected data (Davidson et al. 2021).

While 2D and 3D ultrasound can easily detect

fetal neck masses and associated polyhydramnios

due to impaired swallowing; an important limita-

tion of these techniques is the inability to directly

visualize the airway, thus limiting assessment of

the degree of airway obstruction. MRI plays a

crucial role in identifying fetuses at a risk of

perinatal asphyxia by assessing the size, location,

and internal characteristics of the mass as well as

the degree of tracheal deviation, compression of

the upper airway and oropharyngeal extension as

shown in Fig. 6.25. The advent of ex-utero

intrapartum life-saving treatment measures in

specialist centers has proved life-saving in fetuses

with a critical airway and is reliant on the com-

plex anatomical detail provided by MRI

(Sepulveda et al. 2012).

MRI volumetry allows precise estimates of

total lung volume, assisting neonatal and pediatric

surgical specialists in planning the prenatal and

postnatal care of babies with congenital diaphrag-

matic hernia, congenital pulmonary airway

malformations and anterior abdominal wall

defects (Davidson et al. 2021).

Medical imaging, including ultrasound, CT,

and MRI, plays a valuable role in the evaluation

of shared anatomy in all types of conjoined twins

(e.g., ventral, dorsal, or lateral union) and is

instrumental in decision-making in terms of pre-

natal planning and managing parental

expectations. Prenatal counseling in these cases

focuses on the expected survivability without and

with separation as well as the identiﬁcation of

additional anomalies affecting one of the con-

joined twins. 3D printed models allow the surgi-

cal team to discuss planes of separation and

devise an approach to the multi-stage separation

procedure.

130 S. Fidvi et al.

Fig. 6.22 (a) 2D ultrasound image of a fetus showing

absence of the cranium (anencephaly) giving a frog eye

appearance (white arrow). (b) 3D image of the fetus

conﬁrming the absent cranium (blue arrow). Reproduced

with open-access permission from Akinmoladun et al.

(2020)

Fig. 6.23 Facial features in a fetus with

holoprosencephaly. (a) 2D ultrasound shows proboscis

and severe midfacial hypoplasia. (b) 3D ultrasound sur-

face rendered image shows proboscis, cyclopia, and

arrhinia. (c) a photograph of the neonate with severe facial

malformations. Reproduced with permission from

Sepulveda et al. (2012)

One example of conjoined twins, craniopagus,

is a form of dorsal conjoined twinning with fusion

at the skull (Fig. 6.26). CT is crucial to evaluate

the degree of calvarial bony fusion and MRI is

vital for assessment of the brain and spinal cord

anatomy (Mehollin-Ray 2018). Furthermore, 3D

visualization and printing technologies can allow

for the separation of these twins at a young age,

6 Advanced 3D Visualization and 3D Printing in Radiology 131

Fig. 6.24 Fetus with a cleft lip at 28 weeks’gestation. (a) 3D ultrasound image, (b) 3D virtual model and (c) physical

3D printed model built using a powder-based system. Reproduced with permission from Werner et al. (2010)

Fig. 6.25 Fetal cervical teratoma case. A fetal neck mass

is clearly depicted with (a) 2D ultrasound and (b) 3D-

ultrasound. Fetal MRI shows the internal characteristics

of the mass, polyhydramnios, and partial compression of

the upper airway (c,d). (e) Photograph of the mass at birth

and (f) at the time of surgery (Reproduced with permission

Sepulveda et al. 2012)

therefore harnessing the regenerative capacity of

the brains (Heuer et al. 2019).

132 S. Fidvi et al.

Fig. 6.26 Craniopagus twins showing (a) CT cross-

section with bone segmentation, (b) MRI cross-section

with brain tissue segmentation. (c) 3D printed skull

models printed with stereolithography (3D Systems,

Rock Hill, SC) showing internal vasculature with letters

indicating proper placement for guides. (d) 3D printed

brain models printed with binder jetting technology (CJP

660, 3D Systems, Rock Hill, SC) as two parts to demon-

strate the intended separation plan with the venous sinus.

(e) 3D-printed brain models shown placed together.

Images courtesy of Nicole Wake, PhD, GE Healthcare

6.15 Extended Reality Technologies

and Potential Future

Applications

Advanced 3D medical imaging is used to support

a wide range of surgical operations and for moni-

toring disease progression. Combining 3D imag-

ing methods with the use of new technologies

such as 3D printing can help improve patient

outcomes by reducing operating times, enhancing

precision, and decreasing risk. In addition to 3D

printing, other advanced technologies such as

extended reality can be used to better visualize

medical imaging data. Extended reality is a term

used to describe all real and virtual combined

environments with human-machine interactions

that are generated by computer technologies.

Regarding these technologies, the most popular

are Virtual Reality (VR) and Augmented Reality

(AR). VR is the use of computer technology to

create a completely immersive simulation of a 3D

environment that can be interacted with in a real-

istic, physical method by a person using hand

controllers (Heilig 1960). In contrast, AR uses

computer technology to place virtual 3D objects

within the real environment in the form of an

overlay (Sutherland 1968).

Improved image visualization has been made

possible with advancements of computer power;

however, advanced image post-processing typi-

cally is a laborious time-consuming process.

Automated workﬂows that utilize machine

learning technologies are expected to reduce the

time required to create this 3D content and to

improve the accuracy, thereby allowing 3D

modeling to be performed and implemented more

routinely.

6 Advanced 3D Visualization and 3D Printing in Radiology 133

In the realm of 3D printing, there is ongoing

research into the clinical applications of 3D print-

ing of biomaterials. For example, animal studies

involving 3D-printed biocompatible scaffolds

demonstrate encouraging results for tracheal

reconstruction (Park et al. 2015; Chang et al.

2014; Goldstein et al. 2015). Studies including

customized tympanic membrane printing with

improved resistance compared to the temporalis

fascia and customized septal buttons for septal

perforations with superior compliance and effec-

tiveness have been published (Kozin et al. 2016;

Onerci Altunay et al. 2016). For the treatment of

intervertebral disc degeneration, pre-clinical stud-

ies are currently ongoing to 3D print an elastic

scaffold and deposit substrate/cells to form a

regenerated intervertebral disc as an alternative

to spinal fusion or artiﬁcial disc replacement sur-

gery (Whatley et al. 2011). Combining advanced

radiological imaging methods with bioprinting

has huge potential for advancing personalized

medicine and positively impacting patient care.

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3D printing can be used in spinal surgery

An understanding of relevant anatomy and

3D Visualisation of the Spine 7

Scarlett O’Brien and Nagy Darwish

Abstract

The 3D visualisation of the spine is thought of

from multiple viewpoints. Firstly, radiological

imaging is considered, with plain radiography,

CT and MRI imaging discussed in detail with

relevant applications to spinal surgery.

with multiple applications including educa-

tion, pre-operative planning for complex

cases and making patient-speciﬁc guides and

implants. The rapidly growing ﬁeld of

intraoperative navigation and robotics have

been discussed, in addition to their beneﬁts

and limitations within spinal surgery, as well

as some technical tips.

biomechanics is necessary for any surgeon,

and so this chapter describes the key concepts

to be familiar with, particularly the spinal

motion segment and the different methods for

classifying spinal injuries and how that relates

to stability. The concepts discussed have been

brought together by applying this knowledge

to some interesting clinical cases. They high-

light the importance of 3D visualisation of the

spine, which must be considered throughout

the decision-making process when managing

patients. Spinal surgeons use multiple imaging

modalities, knowledge of anatomy and biome-

chanics, as well as considering the need for

navigation in more complex cases, all on a

daily basis. With the advancement of technol-

ogy available for 3D visualisation of the spine,

we will be able to improve patient outcomes

even further in the future.

S. O’Brien · N. Darwish (✉)

Spinal Trauma Centre, Royal Victoria Hospital, Belfast,

Queen’s University, Belfast, UK

#The Author(s), under exclusive license to Springer Nature Switzerland AG 2023

E. Abdel Meguid et al. (eds.), Biomedical Visualisation, Advances in Experimental Medicine and

Biology 1406, https://doi.org/10.1007/978-3-031-26462-7_7

139

Keywords

Spine · Three-dimensional · Cross-sectional

imaging · Navigation · Biomechanics

7.1 Introduction

Signiﬁcant developments in technology have

enabled us to visualise the details of the human

spine anatomy and abnormalities.

W.C. Rontgen, professor of physics in

Wurzburg, Bavaria, reported the discovery of

the X-ray in 1896 (Röntgen 1896), which is still

essential today for the evaluation and manage-

ment of the spine. Computed tomography

(CT) was the ﬁrst 3D radiological imaging,

which provided more information than plain

radiographs, allowing further classiﬁcation of

injury patterns and pathology of the spine. This

was invented by Sir Godfrey Hounsﬁeld (1980)at

the EMI central research laboratories in 1967, and

the ﬁrst CT scan was performed in Wimbledon,

England.

140 S. O’Brien and N. Darwish

Ten years later, moving away from radiation

technology, Raymond Vahan Damadian, an

American physician, was the ﬁrst inventor of the

MR (Magnetic Resonance) scanner (Young

2004). This revolutionary discovery enabled us

to visualise not only bony skeletal structures but

also to visualise in detail the soft tissues includ-

ing, ligaments, neural and discal structures in all

planes (axial, coronal and sagittal).

3D printing technology allows us to produce

signature models for individual spines. Surgeons

can now design a prosthetic that ﬁts perfectly in

the patient’s body, Dr. Ralph Mobbs, a

neurosurgeon in Sydney, Australia, was the ﬁrst

surgeon to implant a 3D-printed spinal implant to

replace a tumour in the cervical spine and thus

restore stability to the patient’s spine.

Currently, navigation-assisted spine surgery

technology allows the surgeon to access three-

dimensional (3D) and virtual images of the

spine in relation to the surgical instruments

intraoperatively. The Human–Computer Interac-

tion (HCI) concept improves the accuracy of

dealing with the human spine. Robotic spine is

the future; it has expanded the horizon of spine

surgery. It is accurate, safe and minimally

invasive.

This chapter will provide the current

experiences of the authors as spinal orthopaedic

surgeons, combined with a review of the litera-

ture. When discussing spinal patients, we mean

any patient with pathology related to their spine at

any level. This could be in the form of trauma,

such as fractures, ligamentous injuries or spinal

cord injuries, or atraumatic pathology such as

degenerative disease, tumours and infections.

This chapter has been divided into ﬁve

sections: Radiological Visualisation, 3D printing,

Navigation and Robotics, Biomechanics, Clinical

Cases.

7.1.1 Radiological Visualisation

Multiple imaging modalities are currently used to

aid in the management of spinal patients. Radio-

logical imaging has been available since the late

nineteenth century. Imaging now encompasses

both two-dimensional and three-dimensional

scanning, with huge advances since the ﬁrst

X-rays were used. Commonly used imaging in

spinal surgery includes plain ﬁlm radiographs,

computed tomography and magnetic resonance.

Each modality may be used for different reasons

in different situations, with each having a range of

beneﬁts, as well as risks and contraindications. In

this section, each modality will be considered in

detail, including the origins, modes of action and

how we use each modality every day in spinal

surgery.

7.2 Radiography

7.2.1 Background

Wilhelm Roentgen, a physics professor in

Bavaria, ﬁrst discovered the X-ray accidentally

in 1895 (Rontgen 1896). At the time, he was

testing cathode rays, trying to identify if they

could pass through glass. A cathode ray is a

beam of electrons in a vacuum tube travelling

from the negatively charged electrode (cathode)

at one end to the positively charged electrode

(anode) at the other, across a voltage difference

between the electrodes. During his experiment, he

noticed that the rays were able to leak out through

the cathode tube and emit a ﬂuorescent glow on a

nearby screen. He was unsure what this ﬂuores-

cent ray was, and so termed it ‘x-ray’, with ‘x’

meaning unknown. The rays themselves are

termed X-rays, whilst the images the rays produce

when projected onto a screen are termed

radiographs. The ﬁrst radiograph taken was of

Professor Roentgen’s wife’s hand (Fig. 7.1).

Roentgen went on to win the Nobel Prize for

Physics in 1901 for his discovery.

The medical community was quick to realise

the potential for radiographs to be used in patients

to identify fractures or locate bullets or shrapnel

without having to open the body. Within a few

years, the use of X-ray for making diagnoses was

widespread in the United States and Europe. By

1900, radiographs were considered an essential

element in patient care, particularly for their

ability to identify foreign bodies and fractures

(Stimson 1899).

7 3D Visualisation of the Spine 141

Fig. 7.1 Professor Roentgen’sﬁrst radiograph (Baumrind

2011)

7.2.2 Mechanism of Action

A radiograph is produced when a negatively

charged electrode is heated by electricity and

electrons are released, therefore producing

energy. That energy is directed towards a plate,

or anode, at high speed. A radiograph is produced

when the energy collides with the atoms in the

metal plate. When X-rays meet the detector and

create an image, there are ﬁve main densities that

can be visualised; air, fat, soft tissue or ﬂuid,

calcium or bone, and metal. They are a direct

result of how many X-rays have passed through

the subject and arrived at the detector. For exam-

ple, when passing through air, all the X-rays will

continue on to the plate, and none will be

absorbed by the air. This means that the area on

the radiograph will be black as there is no density

to it. Metal, on the other hand, is very dense, and

so almost no X-rays will pass through it and onto

the anode. This means that metal is white on a

radiograph. The densities in between air and

metal vary in how they appear and will be various

shades of grey. Whilst original radiographs were

printed onto ﬁlms and displayed on light boxes,

electronic systems are now used to display the

radiograph on computer screens which allows for

higher-deﬁnition images that we can review in

more detail (Fig. 7.2).

7.2.3 Use in Spinal Surgery

For several spinal conditions, plain radiographs

are detailed enough to make a diagnosis, and no

more advanced imaging is required. A good

example is simple osteoporotic fractures of the

vertebrae, where the fracture is low energy and

unlikely to be unstable (Fig. 7.3). Repeated

radiographs can be taken over time to assess for

further compression or development of kyphosis.

Commonly in practice, we will repeat the radio-

graph when the patient is weight-bearing to assess

for further compression of the vertebrae.

Another example of simple radiography is

identifying and monitoring scoliosis (Fig. 7.4).

Over time, the patient’s spinal curvature may

worsen or change, and various measurements

can be performed on a radiograph to quantify

this. One such measurement is the Cobb angle,

which is deﬁned as the greatest angle at a particu-

lar region of the vertebral column, when

measured from the superior endplate of a superior

vertebra to the inferior endplate of an inferior

vertebra, and refers to angles in the coronal

plane (Coley 2013). An angle of >10 degrees is

highly suggestive of scoliosis, with increasing

angles making intervention or surgery more likely

to be required.

7.2.4 Intraoperative X-Ray

C-Arm systems are used regularly in spinal sur-

gery. C-Arms are ﬂuoroscopy machines which

use image intensiﬁers. This allows high-

resolution X-ray images to be taken and displayed

on a monitor in real time (Fig. 7.5). Modern

C-arms can perform 12 distinct motions, allowing

for the acquisition of almost any radiographic

view (Pally and Kreder 2013). Their name is

derived from their ‘C’shape. This shape allows

the machine to move around the body easily in

order to achieve orthogonal views. They are used

widely in orthopaedic surgery (Ojodu et al. 2018).

The major advantages of intraoperative imaging

are quick and easy identiﬁcation of operative

level, monitoring fracture reduction in multiple

planes and conﬁrming appropriate implant posi-

tion, meaning any errors can be corrected whilst

the patient is still in theatre. It does, however,

have the disadvantage of being quite operator

dependent. It can be difﬁcult to get true AP or

lateral images due to the mobile nature of the

C-Arm, meaning it can be difﬁcult to interpret

the images. There is also the radiation risk to

both the patient and the surgical team, meaning

lead must be worn to protect those near the

C-Arm. There is also potential to de-sterilise the

operative ﬁeld with the C-arm (Biswas et al.

2008).

142 S. O’Brien and N. Darwish

Fig. 7.2 Normal Anterior-

posterior (AP) and lateral

radiograph of cervical spine

Fig. 7.3 Vertebral fracture

pre- and post-operatively

When performing spinal surgery,

intraoperative X-ray is most commonly used to

determine the correct vertebral level. For exam-

ple, when performing an anterior cervical

discectomy and fusion (ACDF), the C-Arm will

be used prior to incision to ensure that the correct

level is chosen to be operated on. The level from

the intraoperative X-ray will be compared to the

level identiﬁed on the MRI scan, which will also

be displayed in theatre. Use of MRI scanning in

spinal surgery will be covered in detail later. It

will also be used during the procedure to conﬁrm

the correct level has been chosen, and this detail

will always be recorded in the operation note.

Images are also often taken when screws and

plates are inserted, to conﬁrm correct positioning

within bone. For any procedure where an implant

of any kind is inserted, a post-operative radio-

graphic check will be taken to compare the

intraoperative position to current position, again

ensuring the implant is positioned correctly.

7 3D Visualisation of the Spine 143

Fig. 7.4 Scoliosis case,

pre- and post-operatively

Fig. 7.5 Intraoperative

C-Arm in use

7.3 Computed Tomography (CT)

7.3.1 Background

CT uses multiple X-ray images stacked together

to develop a three-dimensional image. CT was

ﬁrst developed by Sir Godfrey Hounsﬁeld in

1967, using theoretical calculations developed by

Allan McLeod Cormack (Hounsﬁeld 1980).

Hounsﬁeld was an engineer and inventor, whilst

McLeod Cormack was a physicist. They aimed to

develop X-ray technology, to create a three-

dimensional image which would look inside an

object without opening it. The ﬁrst CT scan was

performed in 1971 in London. The ﬁrst scan was

a CT brain performed in Atkinson Morley’s Hos-

pital in October 1971 (Fig. 7.6). They went on to

win the Nobel prize in Medicine for their inven-

tion in 1979. The early CT scans took 30 minutes

to perform, and several hours to process and

reconstruct the image, often having to be done at

another site. CT is now widely available in almost

all hospitals in the UK. A CT cervical spine can

be performed in around 5 minutes, and images are

uploaded to the imaging system for review within

a few minutes. CT is highly accurate in trauma.

Ninetey-eight percent of cervical spinal injuries

can be detected with CT, compared to radiogra-

phy missing up to 57% of injuries (Jimenez et al.

2008). The major disadvantage of CT compared

to radiography is radiation exposure. A CT of

cervical spine has 90 times more radiation expo-

sure than a series of radiographs (Jimenez et al.

2008).

144 S. O’Brien and N. Darwish

Fig. 7.6 The ﬁrst CT Brain (Maier et al. 2018)

7.3.2 Mechanism of Action

CT uses multiple X-ray images to build up a

cross-sectional image of an area of the body.

Similarly, to plain X-ray, the X-ray beam is emit-

ted from a source, passes through the area of the

body, and then hits a detector. In CT, the source

rotates around the patient, with the detector also

rotating. When each beam is emitted, the detector

will be opposite to the source, and so they move

together. This allows images to be taken from

multiple different angles thus gathering informa-

tion regarding depth.

CT is performed based on different tissues

having different densities. These densities can

be measured from the calculation of the attenua-

tion coefﬁcient. The attenuation coefﬁcient is a

measure of how easily a material can be

penetrated by an incident energy beam.

Hounsﬁeld developed a scale of densities of

tissues with the human body. He used Hounsﬁeld

units (HU) to quantify the density based on four

discreet tissues; air (-1000 HU), fat (-30 to -70

HU), water (0HU) and cortical bone (+1000 HU)

(Kamalian et al. 2016). All tissues are therefore

compared to these densities, and from this, all

materials can be given a density in HU.

7.3.3 Use in Spinal Surgery

7.3.3.1 Trauma

CT is extremely useful in identifying bony

injuries. CT imaging of the spine is carried out

frequently in patients presenting with trauma to

emergency departments. Spinal fractures can

have many patterns and can be due to many

different mechanisms. They can be easily missed

if not investigated. CT is now the modality which

should be used in the ﬁrst instance in most cases

to identify a cervical spine injury, with plain

radiographs rarely used if there is a high level of

suspicion of injury. The National Institute for

Health Care Excellence (NICE) guideline for

head injury recommends many patients with

head injury also have their cervical spine imaged

via CT ((NICE) 2014). The incidence of cervical

spine injury associated with head injury is

between 4% and 8% (Holly et al. 2002), meaning

it is vital that CT cervical spine is performed

alongside CT brain to ensure cervical spine injury

is not missed.

7 3D Visualisation of the Spine 145

In addition to the initial identiﬁcation of frac-

ture, CT allows for fractures to be fully classiﬁed.

This means the exact anatomical position of the

fracture, number of elements or columns involved

and inherently the stability of the injury can be

identiﬁed. This allows surgeons to decide on the

most appropriate management, either operative or

non-operative. If operative management is

indicated, CT imaging allows for operative

planning. CT also identiﬁes if any bony

fragments are present within the spinal canal

(Fig. 7.7) and will prompt clinicians to consider

further imaging with Magnetic Resonance Imag-

ing (MRI) if cord injury is suspected.

7.3.3.2 Tumour

Tumours of the spine can either be benign or

malignant. Malignant tumours are either primary

bone tumours or secondary metastatic disease.

Common metastases arise from renal, lung,

breast, thyroid and prostate primaries. The most

common primary bone malignancy in spine is

multiple myeloma (Kelley et al. 2007). CT can

accurately identify the tumour and give important

information such as size, elements of bone

involved and if stability is compromised. CT is

more sensitive than MRI in determining the

extent of bony destruction, which is useful in

pre-operative planning to determine if operative

intervention is indicated and how it should be

carried out.

Fig. 7.7 Axial CT of the lumbar spine demonstrating

multifragmentary fracture with fragments in the canal

7.3.3.3 CT Myelogram

Myelography is an imaging modality which

utilises intrathecal injection of contrast to delin-

eate spinal conditions. The pathologies

myelography is used to diagnose or monitor

include degenerative spondylosis, nerve root

impingement from any cause and tumours. In

neurosurgery, they are useful for identifying

arachnoid lesions. Prior to the advent of MRI, it

was widely used, both with ﬂuoroscopy and later

CT. The main indication presently is in patients

who have contraindications to MRI, such as an

incompatible pacemaker or spinal stimulator but

require cross-sectional imaging of their spine and

especially thecal sac and contents. Whilst it

provides very useful diagnostic information, it

has the disadvantage of morbidity associated

with radiation and need to inject the subarachnoid

space.

7.4 Magnetic Resonance Imaging

(MRI)

7.4.1 Background

MRI is an imaging modality which creates

images in multiple planes using non-ionising

radiation. Both static and functional imaging can

be produced. Nuclear magnetic resonance

(NMR), later shortened to magnetic resonance,

was discovered in 1945 by Felix Bloch and

Edward Purcell, who were both physicists

(Giunta and Mainz 2020). This principle formed

the basis for MRI scanning. They won the Nobel

prize in Physics in 1952 for this work. In 1969

Dr. Raymond Damadian theorised that cancerous

cells could be detected using NMR, as cancerous

cells hold more water, and would show up in MR

due to increased numbers of hydrogen ions.

Raymond Damadian published the results of his

experiments in 1971.

146 S. O’Brien and N. Darwish

Also, in 1971 Paul Lauterbur theorised a

method using NMR to obtain two-dimensional

and three-dimensional images of living tissue.

The following year, Lauterbur used his NMR

technique to examine two test tubes containing

water. He was able to generate an accurate cross-

sectional image of the test tubes. His paper was

accepted and published by Nature (Lauterbur

1973). Separately, Peter Mansﬁeld published his

research into using magnetic ﬁeld gradients to

create three-dimensional MR images in 1973

(Garroway et al. 1974). Damadian then created

the ﬁrst whole-body human scanner in 1977

(Fig. 7.8). Mansﬁeld and Lauterbur were awarded

the Nobel prize for Medicine in 2003 for their

discoveries regarding magnetic resonance

imaging.

MRI has the advantage of not requiring

ionising radiation, therefore removing this risk

for patients. This means that for patients where

radiation is particularly damaging, such as young

children or pregnant women, MRI is a safe alter-

native. However, given the magnetisation

involved in MRI, it is not suitable for patients

with any indwelling metalwork, such as certain

medical implants. It is also quite a lengthy test,

with a whole spine MRI taking around

45 minutes. This, combined with the noise and

the small space, can make it difﬁcult for some

patients to tolerate.

7.4.2 Mechanism of Action

MRI produces cross-sectional images in any spa-

tial direction; coronal, sagittal, axial, oblique.

NMR relies on the interaction between atomic

nuclei, strong magnetic ﬁelds and radiofrequency

energy. NMR utilises the hydrogen protons

within our bodies, within water, fat and carbohy-

drate molecules. These protons usually spin, cre-

ating a small magnetic charge. When a strong

magnetic ﬁeld is introduced, from the MRI scan-

ner, the protons will align with this ﬁeld. A

radiofrequency pulse is then introduced, which

will disrupt each proton, causing it to rotate

90 or 180 degrees. When the pulse is switched

off, the protons will then return to their natural

position, and this process releases electromag-

netic energy. Differing tissues will release this

energy at different rates, through varying relaxa-

tion processes. The receiving coil then measures

the energy signal coming back from the protons.

This signal then goes to the computer, which uses

a formula to convert the signal to an image

(Berger 2002).

When identifying spinal pathology, three

sequences are commonly used in the MRI scan;

T1 weighted, T2 weighted and Short tau inver-

sion recovery (STIR). The timing of

radiofrequency pulse sequences used to make

T1 images results in images which highlight fat

tissue within the body. The timing of

radiofrequency pulse sequences used to make

T2 images results in images which highlight fat

and water. The timing of the pulse sequence used

to make STIR sequences acts to suppress signal

coming from fatty tissues, and so only water is

highlighted (Patel et al. 2015) (Fig. 7.9).

7.4.3 Use in Spinal Surgery

7.4.3.1 Trauma

Vertebral fractures are increasingly common,

with vertebral compression fractures being the

second most common osteoporotic fracture

(Svensson et al. 2016). It can be unclear if a

fracture is acute or chronic, and patients may

have multilevel fractures. As vertebral compres-

sion fractures are so common, patients presenting

with back pain may have had previous imaging;

either plain radiograph or CT. MRI can be useful

in determining if the fracture is acute or chronic,

as an acute fracture will be bright on the STIR

sequence. It is also useful in determining the

extent of fracture, for example, if it had been

previously unclear if the posterior elements were

involved or not. MRI is the only modality which

will determine if any ligamentous instability has

occurred, particularly of the anterior or posterior

longitudinal ligaments. Ligamentous tear or rup-

ture, combined with some fracture patterns, will

make the spinal construct unstable (Fig. 7.10).

The concept of differing fracture patterns and

their relative stability is discussed later in the

biomechanics section. The major indication for

MRI scanning in a spinal fracture is in patients

with neurological deﬁcits on examination. This

usually indicates a spinal cord, cauda equina or

spinal nerve root injury caused by the fracture.

There are also many spinal cord injuries, which

may not be associated with a fracture, such as

central cord syndrome. These will also require

an urgent MRI to make the diagnosis and guide

management.

7 3D Visualisation of the Spine 147

Fig. 7.8 Dr. Raymond

Damadian’s MRI Scanner

1977 (Winans 2018)

Fig. 7.9 T1/T2/STIR

Sagittal images of lumbar

spine to show comparison

7.4.3.2 Degenerative Disease

One of the most common presentations to the

spinal outpatient department is chronic neck or

back pain, often associated with radiculopathy or

another neurological deﬁcit. If atraumatic, or if a

fracture is excluded on plain imaging, degenera-

tive disease is likely. This is often attributable to

spondylosis (degenerative disc disease)

(Fig. 7.11), spondylolisthesis (degenerative slip),

facet joint hypertrophy or ligamentous hypertro-

phy. All these conditions have the potential to

irritate nerve roots or the spinal cord and cause

associated symptoms. The best way to image

these conditions is with MRI.

148 S. O’Brien and N. Darwish

Fig. 7.10 Anterior longitudinal ligament injury with

associated fracture

7.4.3.3 Tumour

As previously mentioned in the section,

discussing CT scans, malignant tumours in or

around the spine are commonly seen in the ter-

tiary centre. If the diagnosis is uncertain, for

example, it may be difﬁcult to differentiate

between infection and tumour; therefore, MRI

scanning can provide further information to aid

the diagnosis. MRI is useful in classifying the

tumour as extradural, intradural-extramedullary

and intramedullary (Dasarju et al. 2020). This

helps to determine management. When a known

tumour is associated with neurological compro-

mise, metastatic spinal cord compression

(MSCC) should be considered. MSCC is a condi-

tion requiring urgent management, and so access

to MRI for diagnosis should be available 24 hours

a day (NICE 2008).

Fig. 7.11 Multilevel

degenerative disease of

lumbar and cervical spine

7.4.3.4 Cauda Equina Syndrome (CES)

CES is a relatively rare but disabling condition

which can result in motor and sensory deﬁcits,

incontinence of urine and faeces, and loss of

sexual function. Any patient with a possible diag-

nosis of threatened/partial/complete CES requires

urgent investigation. MRI scanning must be

undertaken in an emergency in the patient’s

local hospital and should take precedence over

routine scans ((SBNS) 2018).

7.4.3.5 Recent Advances in MRI

A recent development in MRI is the use of zero

echo time (ET) pulse sequences. A major difﬁ-

culty with traditional MRI scanning is the loud

noise generated from the rapidly switching

currents in the magnetic ﬁeld gradient coils.

Zero ET pulse sequences minimise gradient

switching, and so, almost eliminate noise

completely (Ljungberg et al. 2021). The

sequences required are also shorter, and so scans

can be more efﬁcient. Whilst this technology is

not yet readily available, it will be interesting to

see what developments this advance in MRI will

bring to spinal surgery.

7 3D Visualisation of the Spine 149

7.5 Conclusion

Radiological imaging has advanced exponentially

over the past century. Easy access to multiple

imaging modalities is now available, which

enhances the ability to visualise the human spine

in three dimensions. Within spinal surgery, we

use imaging to make a diagnosis, monitor a con-

dition, make decisions about patient management

and to guide our decision-making

intraoperatively. As discussed later in the chapter,

the future will involve using CT intraoperatively

to navigate surgery, as a further advancement

from intraoperative radiographs.

7.5.1 3D Printing

3D printing is also called additive manufacturing

or rapid prototyping. It was ﬁrst developed by

Charles Hull in the 1980s (Hull 1986), when he

patented the stereolithography device. The tech-

nique uses multiple 2D images, very similar to the

axial cross-sectional images we would be familiar

with from CT and MRI, and as the 2D images are

laid down one on top of the other, a 3D image is

then created.

There are now several different methods for

creating 3D-printed items, but all follow this

basic technique. The three methods currently in

use are: fused deposition modelling (FDM),

stereolithography (SLA) and selective laser

sintering (SLS). FDM uses a heated polymer

that is sequentially layered with a computer extru-

sion nozzle. It is widely used commercially, as it

is relatively low cost and can be done quickly.

However, the products tend not to be stable under

signiﬁcant heat, making them difﬁcult to sterilise,

and therefore not used in surgery. SLA uses light-

curable resin to sequentially add layers. The resin

then undergoes photopolymerisation, where it is

hardened by exposure to a light source. SLS uses

a focused energy source such as an electron beam

or laser to sinter ﬁne powder, in other words, to

convert powder particles into a solid construct

sequentially. Both SLA and SLS can withstand

heat, and thus can be sterilised, making them

options for surgical implants.

7.5.1.1 Use in Spinal Surgery (Sheha

et al. 2019)

Pre-operative

planning

3D models can be printed from

patients imaging. A 3D model of

unusual anatomy or more complex

pathology, allows surgeons to

visualise their plan for surgery.

Intraoperative

guides

Patient-speciﬁc drill guides and

templates can be printed, allowing for

more accurate screw placement.

Patient-speciﬁc

implants

In cases where there is to be a large

resection due to a large tumour,

infection or signiﬁcant trauma, an

implant will need to be ﬁtted to ﬁll the

gap where the resection has been

taken. A speciﬁc implant allows a

more anatomical ﬁt to the patient and

requires a less-extensive dissection,

maintaining the structural integrity of

their spine.

Education At the most basic level, printed

models can demonstrate anatomy and

pathology in 3D, making it easier for

students to visualise and understand.

For surgical trainees, models can be

printed to be used as surgical

simulators. Trainees can learn

techniques on the models, before

transferring them to the operating

theatre.

Many units have access to a 3D printer, which

is connected to the CT imaging system. This

means rapid access to live 3D printing, which

can be used to plan for cases of severe deformity

or complex fractures (Fig. 7.12). These models

are used to visualise the pathology and determine

the exact operative intervention required.

150 S. O’Brien and N. Darwish

Fig. 7.12 3D printed

deformity case in

thoracolumbar spine and

post-operative posterior

stabilisation which have

been printed in our unit

7.5.2 Navigation and Robotics

7.5.2.1 Basics of Navigation

and Robot-Assisted Surgery

Robotic surgery has become commonplace in

many surgical specialties including spinal sur-

gery, general surgery, gynaecology and urology.

Robotic-assisted surgery has also become more

widespread within orthopaedic surgery, particu-

larly in knee arthroplasty. Robotic surgery

systems can either be autonomous, where once

set up, the robot performs most of the operation,

or haptic, where the surgeon is required through-

out the procedure to steer the robot to carry out

the tasks.

7.5.2.2 Use within Spinal Surgery

Within spinal surgery, robotic surgery has been

useful in the insertion of pedicle screws

(Fig. 7.13), which are required for most spinal

stabilisation and fusion procedures. The use of

computer-aided navigation in spinal surgery was

ﬁrst reported by Nolte et al. (1995) where they

used a combination of pre- and intraoperative

information to insert pedicle screws more accu-

rately in an open procedure. Improving the efﬁ-

ciency and accuracy of pedicle screw placement

has many advantages for patients. Screw

malposition can lead to signiﬁcant neurovascular

complications, with so many important structures

in and around the spinal column. Potential

complications include dural tearing, neural dam-

age and vascular or visceral complications. Many

complications lead to serious morbidity and can

necessitate return to theatre, with further

anaesthetic complications and increased risk of

systemic problems. A meta-analysis (Tang et al.

2014) suggests that complication rates and screw

accuracy can be improved by intraoperative navi-

gation techniques.

The ExcelsiusGPS system (Fig. 7.14) (Globus

Medical; Audobon, PA, USA) is a robotic system

Fig. 7.13 Usual position for pedicle screw placement

in use in some UK centres. This system requires a

pre-operative CT scan. Screw position will be

planned from this imaging (Fig. 7.15). The patient

is positioned and prepped as usual. The dynamic

reference base arrays and surveillance markers

are positioned in the bone.

7 3D Visualisation of the Spine 151

Fig. 7.14 The ExcelsiusGPS system

An intraoperative CT using O-arm (Fig. 7.16)

(Medtronic; Dublin, Ireland) is then performed,

which is compared to their pre-operative scan.

Planned screw position will now be conﬁrmed

and any adjustments made.

Small incisions are made, ensuring soft tissues

were adequately dissected. The robotic end effec-

tor arm then moves into position to guide all

movements along this planned trajectory. A pilot

hole is made, then the hole is drilled and tapped,

under navigation ensuring the correct trajectory is

maintained. The screw is ﬁnally positioned, and

placement is conﬁrmed by the robot to match the

planned position. The surgeon receives

immediate feedback that the screw is positioned

correctly.

Whilst additional equipment is required, with

more moving components, there has not been an

increase in surgical site infections associated with

intraoperative navigation. A single-unit study

found no signiﬁcant difference in cases where

no imaging, C-arm ﬂuoroscopy or O-arm

intraoperative CT were used (Kumagai et al.

2022). It was also initially felt that using naviga-

tion would increase surgical times. However,

Khanna et al. (2016) found that whilst operative

times were longer when navigation was ﬁrst

introduced to a unit, over time, operative time

was actually decreased when the surgical team

became more familiar with the equipment. As

navigation becomes more commonplace, it

should also become more efﬁcient than current

ﬂuoroscopic or freehand screw placement.

Advantages and Disadvantages of Robotic

Spinal Surgery (Alluri et al. 2021).

152 S. O’Brien and N. Darwish

Fig. 7.15 Examples of CT navigation images in the thoracolumbar spine

Fig. 7.16 O-arm intraoperative CT

Advantages Disadvantages

Improved clinical

outcomes and reduced

complications

Increased training

required for the surgical

team (Fig. 7.17)

Increased pedicle screw

placement accuracy

Increased cost of

equipment and software

systems

Avoidance of breach of

spinal canal, and so

reduced risk of

development of adjacent

segment disease and spinal

cord damage

Operative time not

decreased until surgeons

and team become familiar

with system –Can take

signiﬁcant time to become

proﬁcient

Reduced radiation risk

when compared to

ﬂuoroscopic guided

instrumentation techniques

Technical issues can be

associated with equipment

or software

Reduced operative time

and increased efﬁciency

7 3D Visualisation of the Spine 153

Fig. 7.17 Theatre team

members using simulation

to train in robotic-assisted

navigation

Tips to Improve Spinal Navigation Surgery

(Cawley et al. 2020)

1. The patient should be positioned prone on the

Jackson table with arms abducted and elbows

ﬂexed. Ensure enough clearance below table

for the navigation system.

2. It is important to dissect only what is

required, as excessive dissection will alter

spinal ﬂexibility and therefore position,

which will make navigation less accurate.

3. The reference frame must be placed solidly in

position and protected throughout the case.

Ensure the frame is not unnecessarily

obscured by drapes.

4. Ensure the drill guide is held ﬁrmly, as any

movements of this will alter the trajectory

when drilling.

5. When drilling, power instruments should be

used to increase accuracy and efﬁciency.

6. The starting point for each drill hole should

ﬁrst be made by a burr or serrated drill guide

to help anchor the drill and prevent slippage,

in turn protecting the trajectory. The naviga-

tion system allows a more lateral entry point

for screw insertion, which allows a longer

trajectory and so a broader construct triangu-

lation. This in turn increases the pull-out

strength of the screw.

7. The templating intraoperatively is carried out

by the circulating surgical team. This

increases the entire team’s awareness of the

need to accurately save the templated image

and communicate the screw size clearly.

8. Using a wire and a cannulated screw tech-

nique is useful, particularly when achieving

the correct entry point is more difﬁcult.

9. When inserting the screw, it is vital to keep

reviewing the images on screen to prevent

over-tightening or perforation of the far cor-

tex. This minimises the risk of nerve root

irritation.

10. Navigation can also be used to deliver intra-

thecal analgesia, which is usually done free-

hand by the anaesthetist. The navigated

method is more accurate, with lower

complications.

7.5.3 Biomechanics

The spine has multiple functions. It supports the

body by providing a structure that loads can be

transmitted through, it allows the spinal cord and

neural structures to be protected, and it allows

motion of the body in multiple 3D planes.

154 S. O’Brien and N. Darwish

Fig. 7.18 Cervical, thoracic and lumbar vertebrae

7.5.3.1 Spinal Anatomy

The spinal column consists of 7 cervical, 12 tho-

racic, 5 lumbar, 5 fused sacral and 5 fused coccy-

geal vertebrae. In the sagittal plane, these are

arranged with four curves; cervical lordosis, tho-

racic kyphosis, lumbar lordosis and

sacrococcygeal kyphosis (sagittal balance). A

typical vertebra contains common features; verte-

bral body, facet joints, spinous and transverse

processes; however, the overall shape varies

widely between different spinal segments

(Fig. 7.18).

7.6 Vertebral Body

The vertebral body is the main load-bearing com-

ponent of the spine. The main force requiring

opposition is compression, which comes from

the weight of the body above the vertebrae. The

structure of the vertebral body allows axial com-

pressive forces to be converted into transverse

tensile forces. The central portion is cancellous

bone, which is surrounded by cortical bone. The

trabecular pattern of the cancellous bone allows

the compressive forces to be distributed through-

out the area evenly. The bone is arranged in

vertical columns and horizontal beams. Their

structure allows the vertebral body to be light-

weight enough to be carried easily, whilst strong

enough to withstand signiﬁcant force. Increasing

the number of columns and beams will propor-

tionally increase the tensile strength of the unit. A

solid structure would be less able to withstand the

load as the forces could not be distributed as

easily.

7.7 Facet Joints

Facet joints make up part of the posterior

elements of the spinal column and allow motion

between vertebrae as well as increasing spinal

stability. They consist of the superior and inferior

articular processes of the superior and inferior

laminae. Different segments of the spinal column

move in slightly different planes as the inclination

of the joint and shape of the joints change. The

cervical spine has a lot of movement, especially

rotation between C1 and C2, as well as ﬂexion,

extension and lateral ﬂexion. The thoracic spine

also has rotation and lateral ﬂexion, but minimal

ﬂexion and extension. In the lumbar spine, the

superior facets are concave and the inferior facets

are convex, allowing signiﬁcant ﬂexion, exten-

sion and lateral ﬂexion but almost no rotation.

They also have a load-bearing function and can

carry up to 30% of the load in combination with

the intervertebral discs.

7.8 Intervertebral Discs

The intervertebral disc consists of a central, gelat-

inous nucleus pulposus and a peripheral annulus

ﬁbrosis. Annulus ﬁbrosis consists of concentric

layers of collagen ﬁbres. The ﬁbres are arranged

30°to the vertical in alternating directions. The

nucleus pulposus is mostly water and generates

hydrostatic pressure. This hydrostatic pressure

exerts tensile stresses in the annulus ﬁbrosis.

There are also the superior and inferior vertebral

end plates which are made up of hyaline cartilage,

and connect the disc to the vertebral body.

The discs are avascular structures, and so rely

on the end plate blood supply for their nutrients.

As the disc is loaded, the hydrostatic pressure

increases, exerting tension on the annulus ﬁbro-

sis, increasing the disc stiffness. When the disc is

loaded the ﬁbres of the annulus ﬁbrosis ﬂatten,

which produces circumferential hoop stresses.

7 3D Visualisation of the Spine 155

7.9 Spinal Ligaments

From anterior to posterior, there are ﬁve main

ligaments associated with the vertebral column:

anterior longitudinal ligament anterior to the ver-

tebral body, posterior longitudinal ligament pos-

terior to the vertebral body, ligamentum ﬂavum

between laminae of adjacent vertebrae,

interspinal ligament between the spinous pro-

cesses and supraspinous ligament posterior to

the spinous processes. The main function of

these ligaments is to provide support to the spinal

column by converting axial loads into tension.

They allow protection of the spinal cord by

restricting the movement of the spinal motion

segment and by absorbing some of the forces.

Conversely, they also permit motion and help

maintain the correct orientation of the vertebral

column.

7.10 Spinous and Transverse

Processes

These bony protrusions provide areas for muscles

and ligaments to attach. They do not offer much

support in terms of load bearing or stability of

the unit.

7.10.1 Spinal Motion Segments

The spine works in multiple functional units

which allow motion between vertebral levels.

Each motion segment consists of two vertebrae

and the disc between them (Fig. 7.19). The inter-

vertebral disc allows ﬂexibility in the unit.

Motion occurs at both the facet joints and the

intervertebral joints. The planes of movement

are ﬂexion and extension in the sagittal plane,

lateral ﬂexion in the coronal plane and rotation

in the axial plane.

7.10.2 Spinal Column Stability

The spinal column is considered stable if normal

mechanical loading does not cause any signiﬁcant

displacement of the spinal motion segment and

more importantly, causes no change in neurologi-

cal status. This means in practice, that the spinal

column is considered unstable if there is deforma-

tion when a patient axially loads, i.e. stands or sits

upright. The question of stability is one of the

most important things to consider when assessing

a patient with spinal trauma.

Several classiﬁcation systems are available

which help to determine the stability of the

thoracolumbar spine. In 1963, Holdsworth

(1963) published his two-column classiﬁcation

system. In this, he deﬁned the anterior column

to begin at the anterior longitudinal ligament and

ﬁnish at the posterior longitudinal. Elements pos-

terior to the posterior longitudinal ligament are

deemed the posterior column. He deemed that the

posterior ligament complex was vital in determin-

ing stability. If both anterior and posterior

columns were injured, the injury was unstable.

If a single column was injured, the stability

depended on the integrity of the posterior

ligaments, and so the posterior column involve-

ment. The only imaging modality available to

Holdsworth was plain radiography.

This two-column classiﬁcation system has

now largely been superseded by the three-column

classiﬁcation developed by Denis (1983) in the

1980s. With the advent of CT, Denis had more

information available and expanded the columns.

The anterior column now involved the anterior

longitudinal ligament and the anterior half of

vertebral body and intervertebral disc. The middle

column included the posterior half of the vertebral

body, disc and posterior longitudinal ligament.

The posterior column was the same, including

all posterior elements. Single-column injuries

were all deemed stable. Three-column injuries

were unstable. Two-column injuries involving

the middle column are usually unstable.

156 S. O’Brien and N. Darwish

Fig. 7.19 Spinal motion

segment, including two

vertebrae and their

intervertebral disc

More recently we are using the revised AO

classiﬁcation (Vu and Gendelberg 2020). This

system includes three groups of fractures: com-

pression (Type A), distraction (Type B) and tor-

sional injuries (Type C) (Fig. 7.20). Type A

injuries are simple compression injuries, Type B

injuries are due to failure of the anterior or poste-

rior tension band without gross translation, and

Type C injuries are dislocation, rotational or

displacement-type injuries. Within each type,

there are subgroups which describe the severity

and stability of the injury. A higher number

indicates a more complex or unstable injury, for

example, an A4 injury is a complete burst fracture

involving both endplates and the posterior wall,

but with the posterior ligaments intact. It is much

more complex than an A1 injury, which includes

a simple end plate, with no involvement of the

posterior elements. In clinical practice, all A type

fractures and B1 fractures can be managed

non-operatively. B2 can be managed either

non-operatively or operatively, whilst B3 and C

fractures are all unstable and require operative

stabilisation.

Clinical clues to an unstable spine include

increased pain on axial loading, neurological

compromise and signiﬁcant deformation.

7.10.3 Clinical Cases

7.10.3.1 Gunshot

A 35-year-old male presented with a gunshot

wound to his back. He was a usually ﬁt and well

male, with a history of previous substance mis-

use. He sustained a single entry wound at the

level of his lumbar spine. On initial examination,

he had a right-sided neurological deﬁcit. He had

altered sensation at L3-S1, with reduced power

3/5 in this distribution. He also had absent knee

and ankle reﬂexes. There was no associated

bowel or bladder disturbance. Initial imaging

was carried out with a CT trauma scan of head,

cervical spine, chest, abdomen and pelvis

(Fig. 7.21). This identiﬁed a comminuted fracture

of L3, with the bullet lodged in the right pedicle.

The fracture extended through the posterior

elements of L3 as well as involved the L2 spinous

process. Bony fragments were identiﬁed within

the spinal canal at the L2/3 level. Multiple gas

locules were demonstrated tracking from the skin.

Imaging also presented a left grade V renal injury

with extensive perinephric haematoma.

7 3D Visualisation of the Spine 157

Fig. 7.20 Revised AO Classiﬁcation of Spinal Fractures

This gentleman was initially managed with

suitable resuscitation in the emergency depart-

ment. He required blood products, intravenous

ﬂuids and signiﬁcant analgesia. This was treated

as an open injury, which was likely to be

contaminated. As such, he was managed as per

the British Orthopaedic Association (BOA) and

British Association of Plastic and Reconstructive

Surgeons (BAPRAS) guidance on open fractures

(1). He received intravenous antibiotics and a

tetanus vaccine as well as tetanus immunoglobu-

lin. A urinary catheter was inserted, with the

urology team providing input for his renal injury.

He proceeded to the theatre the next morning

for operative intervention for his spinal injury. At

the time of surgery, the fracture was noted in the

L3 pedicle. The bullet was successfully removed

in one piece and sent to police as evidence. The

spine was then stabilised with pedicle screws

L2-L4 and pre-contoured rods. Intraoperative

ﬂuoroscopy (Fig. 7.22) conﬁrmed all screws

were appropriately sited. The gunshot tract was

extensively washed and debrided.

158 S. O’Brien and N. Darwish

Fig. 7.21 Pre-operative coronal, sagittal and axial CT images

Post-operatively the patient made good prog-

ress. His renal injury was managed

non-operatively. A check radiograph was

performed when stable (Fig. 7.23), which showed

pedicle screws and rods to be appropriately posi-

tioned, and good overall lumbar alignment. At

review, his sensation and power had improved,

and he was managing to mobilise independently

without any walking aid.

Diagnosis and operative planning would have

been impossible in this case without 3D radiolog-

ical imaging. His initial trauma scan was

reconstructed into a 3D image which could be

rotated, allowing the bullet to be easily visualised

in relation to his bony anatomy. Intraoperative

imaging was also vital to conﬁrm the correct

level and ensure all metalwork was positioned

accurately.

7.10.3.2 Fracture on Background

of Ankylosing Spondylitis

The next case is that of a 67-year-old male who

was involved in a road trafﬁc collision. He was a

restrained driver, who was hit head-on by another

motorist. He was able to self-extricate and

initially to mobilise at the scene; however, he

did have signiﬁcant thoracic back pain. He has a

history of both ankylosing spondylitis and rheu-

matoid arthritis and was on biological therapy. He

had previous vertebroplasty surgery treating col-

lapse fractures of the thoracic spine, as well as

multiple non-operatively managed thoracic verte-

bral fractures. On examination, he had no focal

neurological deﬁcit. He was haemodynamically

stable. His main complaint was of thoracic back

pain. No other injury was identiﬁed outside his

spine on primary or secondary survey.

Initial imaging was performed with a CT of the

thoracic spine (Fig. 7.24). This identiﬁed a three-

column, unstable T10 fracture on the background

of signiﬁcant ankyloses. He had subsequent

imaging with a CT of the cervical and lumbar

spine to complete the series of CT imaging.

Initially, a fracture through the C6/7 disc space

was queried. At this stage, the patient was trans-

ferred to the tertiary spinal unit and had an MRI

Whole Spine (Fig. 7.25). The injury at C6/7 was

felt to be long-standing on this imaging and no

cord injury was identiﬁed; however, a posterior

epidural haematoma and anterior longitudinal lig-

ament injury were detected. Combining this infor-

mation, the patient was felt to have a highly

unstable T10 fracture and proceeded to theatre

the same day as an emergency.

7 3D Visualisation of the Spine 159

Fig. 7.22 Intraoperative

imaging

Fig. 7.23 Post-operative

radiographs

He had posterior stabilisation of T10 with ped-

icle screws and rods from T7-L2. Levels were

checked pre-operatively, and screw position was

checked intraoperatively using ﬂuoroscopy

(Fig. 7.26).

160 S. O’Brien and N. Darwish

Fig. 7.24 Pre-operative sagittal MRI of cervical and thoracolumbar spine. Demonstrating the T10 fracture, as well as

excluding a new cervical injury

Post-operatively he recovered well. There was

no neurological deﬁcit and he has been able to

mobilise independently. Post-operative

radiographs (Fig. 7.27) obtained showed metal-

work to be in the appropriate position.

This case highlights the importance of

recognising the extensive instability of this frac-

ture pattern in patients with ankylosing spondyli-

tis, and to a lesser extent, diffuse idiopathic

skeletal hyperostosis (DISH). Their spine is sig-

niﬁcantly stiffer than a spinal column with no

underlying condition, and therefore they are

likely to sustain more signiﬁcant injuries with a

higher risk of neurological compromise. It was

vital to obtain completion imaging of the whole

spine, as injuries at other sites are common, with

the incidence being quoted as 13.1%

(Lukasiewicz et al. 2016). With consideration of

all these factors, these injuries should be treated

as an emergency and should have posterior

stabilisation on the same day.

7.10.3.3 Cauda Equina Syndrome

This case is of a 43-year-old male who had a long-

standing history of lower back pain. For many

years he also had radicular pain in his right leg.

He presented to the emergency department with

1 week of increased lower back pain. His

radiculopathy had progressed to bilateral pain

over 3 days. He had 36 hours of difﬁculty with

urination, having difﬁculty initiating a urinary

stream. He was an otherwise ﬁt and healthy gen-

tleman. On examination, his power was intact

throughout all myotomes and sensation intact

throughout all lower limb dermatomes. He did

have altered sensation in the S2–4 dermatomes

in the perianal area; however, anal tone was

intact. On presentation, he had difﬁculty initiating

a urinary stream, and so was catheterised. He was

found to have less than 200 ml residual volume in

his bladder.

An MRI was arranged by the emergency

department, which demonstrated a large L5/S1

disc protrusion which was causing cauda equina

compression (Fig. 7.28). He proceeded to the

theatre the following day for L5/S1 discectomy

and decompression.

7 3D Visualisation of the Spine 161

Fig. 7.25 Sagittal CT of

thoracic spine and axial CT

of T10. Arrow

demonstrates the signiﬁcant

anterior opening

Post-operatively he recovered well. He had no

ongoing neurological deﬁcit and was discharged

on day 3 post-operatively.

This gentleman presented with classical

symptoms of cauda equina syndrome. He had

back pain, bilateral leg pain, urinary disturbance

and perianal sensory disturbance. Excellent emer-

gency access to MRI scanning afforded this gen-

tleman the ability to be managed in a timely

fashion and thus avoided long-term neurological

compromise.

Fig. 7.26 Intraoperative imaging

7.10.3.4 Acute Thoracic Disc

with Neurological Compromise

The next case is that of a 46-year-old male who

presented with thoracic back pain, leg weakness

and loss of bladder control. He had no previous

signiﬁcant medical history. Initially, he was

catheterised, with no signiﬁcant residual volume

and was able to feel the catheter. He had normal

sensation in all lower limb dermatomes and nor-

mal power in all lower limb myotomes.

He proceeded to MRI (Fig. 7.29) which

demonstrated a T10/11disc extrusion with cord

compression.

At this stage, given there was no neurological

deﬁcit, the decision was made to manage this

patient non-operatively. His bladder symptoms

resolved over 48 hours and his catheter was suc-

cessfully removed. Unfortunately, he subse-

quently went into urinary retention and had to

be re-catheterised a few days later. Power and

sensation remained intact in his lower limbs.

Operative management was then felt to be in his

best interest, and so he proceeded to theatre for

posterior decompression of T10/11.

The operation was performed as a dual consul-

tant case and a satisfactory decompression was

achieved, meaning both operating consultants felt

that the spinal cord was completely free with no

pathological tissue indenting it. The level was

conﬁrmed on intraoperative imaging above.

Unfortunately, the patient developed a complete

loss of power in lower limbs bilaterally in recov-

ery. He had a sensory level at L1 and was unable

to feel the catheter. An emergency MRI was

arranged (Fig. 7.30).

162 S. O’Brien and N. Darwish

Fig. 7.27 Post-operative

AP and lateral radiographs

Fig. 7.28 Axial and sagittal MRI of lumbar spine, demonstrating large L5/S1 disc with cauda equina compression

The MRI conﬁrmed that a satisfactory poste-

rior decompression had been performed at the

correct level. There was no evidence of epidural

haematoma, but cord signal change was evident.

However, given the signiﬁcant neurological deﬁ-

cit, the decision was made to proceed to anterior

decompression. Thoracic surgeons gained access

to the chest anteriorly and an anterior hemi

corpectomy, discectomy and cage was performed

(Fig. 7.31).

7 3D Visualisation of the Spine 163

Fig. 7.29 Axial and

sagittal MRI images of

thoracic disc

Post-operatively he was initially managed in

the high-dependency unit; however, he was

discharged to the ward within 48 hours.

His MRI was then repeated one week follow-

ing the initial surgery (Fig. 7.32).

This demonstrated a reduction in cord oedema,

but ongoing disc extrusion. The patient then

proceeded to revision discectomy and posterior

stabilisation T9-T12 (Fig. 7.33).

This patient made little neurological improve-

ment despite intensive intervention. Following

his admission, he had a period of rehabilitation

in the spinal injuries unit where he made some

improvements and was wheelchair independent

with most activities of daily living on his

discharge.

This case discusses thoracic disc disease with

compression of the spinal cord. MRI imaging was

vital for operative planning, and management was

changed by MRI appearances. Our learning point

from this case highlighted that navigation would

be useful in a complex case like this, to minimise

complications and prevent neurological compro-

mise. Another key issue is that time to surgery

should be as soon as possible to ensure the best

outcome.

7.10.3.5 Inflammatory

A 40-year-old female patient presented with a few

weeks of back pain and 4 days of right leg radic-

ular pain. She reported a 5 kg weight loss over

several months. She was systemically well with

no fevers. No previous signiﬁcant past medical

history was noted. On initial examination, she had

right lower limb weakness in the L2/3 myotomes,

but intact distally. Bilateral hyperreﬂexia with

upgoing plantar reﬂexes. No bowel or bladder

disturbance initially. She proceeded to MRI of

whole spine. This demonstrated extensive multi-

level spondylodiscitis with multiple abscesses.

There was almost complete destruction of the T5

vertebrae with associated kyphosis. There was a

collection at the T4-6 level with cord compres-

sion. A similar picture was evident around T12

and L3/4 with bony destruction and ring enhanc-

ing collections (Fig. 7.34).

164 S. O’Brien and N. Darwish

Fig. 7.30 Post-operative

MRI imaging showing cord

signal change

Following MRI, the likely diagnosis was felt

to be tuberculous spondylodiscitis with abscess

formation. The patient was immediately

commenced on an antimicrobial regime to treat

TB and transferred to the spinal unit for

intervention.

Fig. 7.31 Intraoperative imaging demonstrating

thoracic cage

Following transfer, the patient developed

rapid, progressive deterioration, with complete

paralysis of the lower limb. Her sensory level

was at T7. Her power was 2/5 bilaterally in L2-4

and 1/5 in L5-S1. She had been in urinary reten-

tion and was catheterised. She proceeded to the-

atre for posterior decompression and stabilisation

T2 to L2 (Fig. 7.35). Such a signiﬁcant

stabilisation was required due to the signiﬁcant

bony destruction and compression of the spinal

cord. A large volume of pus was evacuated and

these samples conﬁrmed the diagnosis of

TB. Overall spinal alignment was considerably

improved with the correction of the previous

kyphosis.

Post-operatively she remained systemically

well. She had extensive input from infectious

diseases, who monitored her TB therapy. She

completed a period of rehabilitation in the spinal

injuries unit where some improvements were

made. She was continent of bowel and bladder.

She was able to mobilise short distances with a

rollator as power improved.

This patient had extremely extensive tubercu-

lous spondylodiscitis on initial imaging. She had

a very successful decompression with copious

pus drained and a stable ﬁxation performed.

This highlights TB as a highly aggressive

pathogen, and an important one to consider. This

was a complex case which required input from the

whole multidisciplinary team, including spinal

surgeons, infectious diseases physicians and the

specialist rehabilitation team. This case highlights

the need for emergency surgical decompression

with neurological compromise, as this patient had

complete lower limb paralysis pre-operatively

and is now able to mobilise.

7 3D Visualisation of the Spine 165

Fig. 7.32 Post-operative

sagittal and axial MRI

1 week following surgery

Fig. 7.33 Final post-

operative AP and lateral

radiographs

166 S. O’Brien and N. Darwish

Fig. 7.34 Initial Whole

Spine MRI Sagittal and

axial cut at T12

Fig. 7.35 Post-operative

AP and lateral radiographs

7 3D Visualisation of the Spine 167

7.11 Conclusion

Visualisation of the human spine allows us to

better understand and manage patients with spinal

pathology. In this chapter, basic visualisation

techniques were discussed including plain

radiographs, as well as more modern and complex

modalities such as the MRI advance of zero ET

and intraoperative CT using the O-arm. A sound

knowledge of anatomy and spinal biomechanics

is vital to truly comprehend the 3D spine, and this

chapter discusses the basic aspects of these. 3D

printing can be used to enhance spinal surgery in

many ways, including improved pre-operative

planning, use in intraoperative templating and

for improved surgical education. Navigation and

robotic surgery allow surgeons to visualise the

human spine in real-time 3D, therefore allowing

improved screw positioning and intraoperative

feedback. This development of human–computer

interaction has allowed higher levels of surgical

accuracy, and therefore overall improved surgical

outcomes. There is a continuous development of

new technologies, such as robotics and virtual

reality, which will only improve patient care in

the future.

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Part III

Anatomy Education

Visualization in Anatomy Education 8

Apurba Patra, Nagavalli Basavanna Pushpa,

and Kumar Satish Ravi

Abstract

In the post-pandemic era, one of the signiﬁcant

challenges for anatomy teachers is to recipro-

cate the experience of practical exposure while

teaching the subject to undergraduates. These

challenges span from conducting cadaveric

dissections to handling real human bones,

museum specimens, and tissue sections in the

histology lab. Such exposures help the

instructors to develop interactive communica-

tion with their fellow students and thus help to

enhance communication skills among them.

Recently, anatomy teachers all over the world

started using cutting-edge educational

technologies to make teaching-learning

experiences for students more engaging, inter-

esting, and interactive. Utilizing such cutting-

edge educational technologies was an “option”

prior to the pandemic, but the pandemic has

signiﬁcantly altered the situation. What was

previously an “option”is now a “compulsion.”

Despite the fact that the majority of medical

schools have resumed their regular on-campus

classes, body donation and the availability of

cadavers remain extremely limited, resulting

in a deadlock. Anatomy teachers must incor-

porate cutting-edge educational technologies

into their teaching and learning activities to

make the subject more visual. In this chapter,

we have attempted to discuss various new

technologies which can provide a near-

realistic perception of anatomical structures

as a complementary tool for dissection/

cadaver, various visualization techniques cur-

rently available and explore their importance

as a pedagogic alternative in learning anatomy.

We also discussed the recent advancement in

visualization techniques and the pros and cons

of technology-based visualization. This chap-

ter identiﬁes the limitations of technology-

based visualization as a supplement and

discusses effective utilization as an adjunct to

the conventional pedagogical approaches to

undergraduate anatomy education.

A. Patra

Department of Anatomy, All India Institute of Medical

Sciences, Bathinda, Bathinda, Punjab, India

N. B. Pushpa

Department of Anatomy, JSS Medical College,

JSSAHER, Mysore, Karnataka, India

K. S. Ravi (✉)

Department of Anatomy, All India Institute of Medical

Sciences, Rishikesh, Rishikesh, Uttarakhand, India

#The Author(s), under exclusive license to Springer Nature Switzerland AG 2023

E. Abdel Meguid et al. (eds.), Biomedical Visualisation, Advances in Experimental Medicine and

Biology 1406, https://doi.org/10.1007/978-3-031-26462-7_8

171

Keywords

Anatomy education · Curriculum · Cadavers ·

Remote learning · Virtualization

8.1 Introduction

Human anatomy is a fundamental subject in med-

ical education. A basic understanding of the

bodily structure and architecture of humans is a

must to become a competent physician. Further-

more, sound knowledge of regional anatomy is

necessary to know the disease process, gain sur-

gical skills and interpret radiological imaging.

Learning anatomy entails the study of body tissue

and organ system and appreciating their 3D ori-

entation (Basavanna et al. 2022). Although the

learning modality (visual, auditory, reading, and

kinaesthetic) preference of the student has to be

considered while delivering the concepts, it is not

a hard and fast rule to stick to it. Moreover, anat-

omy learning is not limited to understanding the

3D orientation of the organ system but also under-

standing the complex dynamics of development,

the microstructure of tissues, and analyzing and

interpreting radiology images (Hall 2016). The

potential to interpret 3D anatomical features in

2D cross-sectional text or pictures can be trouble-

some for undergraduate students (Keenan and

Ben Awadh 2019).

172 A. Patra et al.

Presently, the main impetus behind

incorporating technology-based learning in the

ongoing curriculum is to enhance undergraduate

medical students’learning of clinically oriented

anatomy, improvement in skill coordination, team

training, and enhancement of perceptual variation

(Guze 2015). Here, we will describe various visu-

alization techniques currently available and

explore their importance as a pedagogic alterna-

tive in learning anatomy. Following this, we will

discuss the recent advancement in visualization

techniques. Lastly, we wish to brieﬂy debate the

merits and demerits of technology-based visuali-

zation and identify their limitations as a supple-

ment and effective tool to utilize as an adjunct to

the conventional pedagogical approaches to

undergraduate anatomy education.

8.1.1 Changing Scenario in Anatomy

Education

For decades, anatomy has been taught and

learned through lectures and textbooks with 2D

or line diagrams. Cadaveric dissection is the most

admired and widespread way of studying anat-

omy, which enables us to visualize structures in

their three-dimensional form with spatial

relationships. Many studies have concluded that

cadaveric dissection is the only 3D experience

students receive in their anatomy learning. The

limited exposure to 3D learning increases the

cognitive burden on students since they struggle

to translate the 2D experience into a 3D experi-

ence (Guillot et al. 2007). Also, observation and

visualization are essential components of learning

anatomy (Jasani and Saks 2013). Not only gross

anatomy but understanding developmental anat-

omy/embryology and complex dynamics is difﬁ-

cult with available handmade models (Ruiz et al.

2009). Over recent years, anatomy teaching has

been moving from conventional cadaver dissec-

tion to virtual cadavers, 3D printed specimens,

and computed 3D body systems (Sharma and

Kumar 2021). The virtual dissection table utilizes

simulation technology to offer a realistic 3D visu-

alization of the anatomical details of a virtual

cadaver. The most signiﬁcant advantage of a vir-

tual cadaver is that students can “do-redo-repeat”

dissection. This can signiﬁcantly aid most medi-

cal schools which do not have access to donors

due to the pandemic, and virtual dissectors can

help to compensate for the deﬁcit (Ravi 2020).

Most medical schools, which used almost no

technology-based medical education applications

in the pre-pandemic era, are now widely adopting

these tools to effectively deliver anatomy

knowledge.

8.1.2 Traditional 3D Visualization

in Anatomy and Its Limitations

The effectiveness and applicability of teaching

depends on the optimal availability of study mate-

rial. In traditional pedagogy, cadaver dissections,

real bones, skeletons, museum specimens, and

handmade clay models have always remained

the primary sources of learning 3D anatomy.

Although all of these modalities are proven

ways of learning anatomy, they have their own

limitations. The lack of cadavers and the ethics

associated with their acquisition discourage the

administration of some medical schools from

continuing with cadaveric dissection. Very

recently, the pandemic has shown us how

important it is for medical schools to be prepared

with suitable alternatives. Real human bones and

articulated skeletons are a good resource for

studying musculoskeletal anatomy and their spa-

tial relationships, but less availability and enor-

mous cost make their acquisition problematic. An

essential part of teaching anatomy is the use of

high-quality human anatomical specimens that

can demonstrate the topographical relationships

of individual anatomical structures. However,

long-term use of these real human specimens

leads to their degradation, which reduces the

quality of teaching. Handmade clay models

depicting different stages of human development

are also a very good resource for understanding

the dynamic events of embryogenesis. However,

the production of these models requires human

precision, and their fragility also leads to degra-

dation, limiting the quality of teaching. Students

also use anatomical literature, scripts,

illustrations, and atlases. However, anatomical

atlases, which are indispensable in learning anat-

omy, are quite expensive for students. This

accounts for the emerging use of 3D interactive

anatomy models as signiﬁcant alternative study

tools for students. These tools help them to

correctly interrogate and understand the really

intricate topographical and functional

relationships of anatomical structures (Brazina

et al. 2014). It is indeed of great concern that

students have opined that they have acquired a

lack of knowledge of anatomy despite having

good quality specimens supported by various

teaching methods (Dev et al. 2002; Basavanna

et al. 2022).

8 Visualization in Anatomy Education 173

8.2 Potential Technological Tools

of Visualization in the Teaching

of Anatomy

With this background, we would like to present

possible solutions for supporting education

through various interactive applications. In par-

ticular, newer applications such as 3D printing,

computerized learning management systems

(LMS), virtual classrooms, artiﬁcial intelligence,

cloud technology, virtual dissection, simulation,

radiological imaging, immersive technology,

web-based learning, and interactive 3D computer

graphics, etc. (Owolabi and Bekele 2021). The

3D approach provides students with an advanced

way to work with scanned images of human

tissues and organs using 3D computer simulation.

Interactive 3D models were developed with the

appropriate modeling tool for research purposes.

Typically, this period is a transition to a visuali-

zation and simulation environment created to

solve a speciﬁc problem or cope with the global

learning environment of current 3D visual tools

(Fig. 8.1).

8.2.1 3D Printing in Anatomy

Three-dimensional printing is a technique of cre-

ating a physical object using a 3D computer

model. Here, printed materials are layered on

top of each other using computer control to pro-

duce an object/pattern that matches the real thing.

Figure 8.2 represents a ﬂow chart showing the

methodology of developing 3D printed models

from a patient CT/MRI. It is another newer

modality to create a 3D model of dissected

specimens so that the complex architecture of

any tissue can be made accessible to students in

a more comprehensive way. 3D printing is an

impressive tool for creating synthetic models of

organs and structures. It is possible to create

models that students can explore, interact with,

and learn from in a group. 3D printing is very

useful for both visual and kinaesthetic learners. It

can reduce the time spent on planning and design-

ing models (Abou Hashem et al. 2015; Sharma

and Kumar 2021). Other advantages include user-

friendly or easy creation of geometric parts with

added complexity, replication of models in large

numbers or easy prototyping, cost-effectiveness

(no molds required), fully customizable, creation

of parts with speciﬁc properties, accuracy

(computer-aided design), no-use of formalin, no

human or animal tissues are used, so there are no

ethical concerns. Apart from that, creating models

with anomalies to arouse interest in applied anat-

omy and clinical cases (Ye et al. 2020). There

were also several disadvantages, including

fragility as the parts are assembled layer over

layer, which decreases the strength by 10–50%,

more cost at high volume, post-processing

requirements such as grinding or smoothing to

create the desired ﬁnish, and heat treatment to

achieve speciﬁc material properties or ﬁnal

processing.

174 A. Patra et al.

Fig. 8.1 Represents a ﬂow chart listing newer tools/techniques for visualization in anatomy other than cadaveric

dissection

Role of 3D Printing Anatomy Teaching

Amongst the various teaching aids available, dis-

section is the elite and most effective teaching

mode. The unavailability of cadaver dissection

has resulted in hindrances in anatomy education

for students (Kerby et al. 2011). The complex

architecture of organs (liver) or small bones (ear

ossicles) may not be clearly visible during

dissection, and students might ﬁnd difﬁculty

understanding them. 3D printed models of such

structures can be of great help. The complex

architecture of extraocular muscles with respect

to the eyeball is quite difﬁcult to understand for

medical students; hence a 3D printed model of

such a structure aids students in understanding the

system effectively (Fig. 8.3). Moreover, 3DP of

organs/bones is a cost-effective way that provides

a haptic study for better understanding (Kong

et al. 2016).

Fig. 8.2 Flow chart

showing the methodology

of developing 3D printed

models from Patient

CT/MRI

3D printed models of the skull, transverse

sections of the brain, orbital anatomy, and cardio-

vascular development (Fig. 8.3) can be used to

teach complex anatomy of difﬁcult regions and

different surgical procedures pertaining to these

regions. Mainly related to the heart, cardiovascu-

lar 3D printing augments the diagnostic work-up

of complex congenital heart defects and assists in

planning surgical and interventional procedures.

8 Visualization in Anatomy Education 175

Fig. 8.3 3D printing models of different organs (owned by authors for copyright)

8.2.2 3D Learning Management

System

Currently, LMS offers limited access for teachers

and students to design teaching and learning

activities based on experience and collaboration.

Integrating two environments (LMS and 3D) is

not the only method to improve learning; this

combination can enhance students’independent

learning skills (Yasar and Adiguzel 2010). 3D

LMS Software provides students with interactive

anatomy resources. This software enables users

with an interactive way to visualize, appreciate,

dissect, and study real-life examples of human

anatomy on their computer screens. Below we

explain the student and Instructor’s 3D views.

Student view:

Students can download the 3D anatomy soft-

ware directly to their PC/laptop and access 3D

anatomy resources anytime, anywhere. 3D

Anatomical Visualization uses CT/MRI scans

from real patients to develop interactive 3D

visualizations that depict realistic patient anat-

omy. The screen’s pan and zoom provisions

allow students to visualize and study any

anatomical structures with their 3D spatial

relationships. Students can peel back layers of

tissue and explore through tissue and color

modes to appreciate and learn each structure

in 3D.

Instructors’View:

With the help of interactive 3D visualizations,

teachers can enhance less engaging lecture

materials to make them more interesting so that

students can better understand anatomical

concepts before entering the dissection lab.

Instructor features in 3D anatomy software make

it easy to develop engaging and real anatomical

material for the lecture. For example, using an

online portal, instructors can easily assign quizzes

and homework to the students and track their

performance with automated grading. 3D visuali-

zation scans reconstructed from real CT/MRI

scans can aid in student interaction, dissection,

and teaching. In addition, teachers can mark and

annotate speciﬁc anatomical structures/including

additional content such as images or text from the

reading material.

176 A. Patra et al.

The multimodal role of 3D LMS in the trans-

formation of anatomy education:

3D LMS has enormous potential to make anat-

omy visible. For this, we need to incorporate and

implement it in the ongoing curriculum using

three formats as suggested below:

Inside the lecture hall: Learning objectives and

course materials can be presented to students with

the help of the lecturer’s version of the software.

Depending on instructor preference, this can be

completed before the session and incorporated

into PowerPoint videos/slides or demonstrated

live in front of students with a multimedia

projector.

Inside the Dissection Lab: Students can work

individually/in small groups to complete the vir-

tual dissection, which is part of the active learning

modules. Every lab module could include step-

by-step instructions for the students to complete a

virtual autopsy.

At Home: Students can be assigned homework

and apply their classroom and lab knowledge to

answer interactive quizzes provided in the assess-

ment modules. In addition, several educational

programs provide ungraded review modules to

help students prepare for exams.

3D LMS provides a new range of learning

opportunities. The nature of the environment

provided is generative, enabling users to explore

and interact with an already existing 3D environ-

ment and extend that experience by creating their

own study materials (Kluge and Riley 2008).

Elsevier’s Complete 3D Anatomy tutorial was

one of the tools which helped teach anatomy

during the pandemic. The software has voice

recording features that allow video creation

using dynamic 3D representations to design

video lectures linked to a learning management

system (LMS) or directly through the software

platform. In addition, this 3D software also offers

a kinesthetic advantage based on the VARK

learning theory (Othmana and Amiruddinb

2010; Bhagat et al. 2015; Mozaffari et al. 2020).

8.2.3 Virtual Classroom

A virtual classroom (VC) is a video conferencing

tool in which instructors/teachers and participants

engage using learning material. The difference

between VC and other video conferencing tools

is that VCs provide add-on features necessary for

a learning environment. This platform aids in

making learning an interactive and engaging pro-

cess in a controlled environment that goes beyond

the classroom. Instructors can access the class-

room prior to the session to prepare study mate-

rial. This study content, as well as the recording

of the session, is available after the class for

reference by both instructors and participants.

Participants can join the virtual classroom

platforms using any device with an internet con-

nection. This type of ﬂexibility allows

participants to learn the content anywhere in the

world. Another great beneﬁt of a virtual class-

room is that it makes it easy to track student

progress. Instructors can collect data such as

class attendance and student activity. They can

track participant progress through online surveys

and analytics, identify areas of difﬁculty, and use

visual tools to facilitate learning challenges for

participants. Finally, many virtual classroom

platforms can be integrated into an institution’s

existing Learning Management System (LMS).

Advanced platforms aid Learning Tools Interop-

erability (LTI) so that the virtual classroom sys-

tem and LMS can communicate with each other,

making the whole perspective more effective.

Current techniques such as smart boards to draw

diagrams, video presentations with hyperlinks

(Osmosis videos (Haynes et al. 2014; Guven

et al. 2020) and ScholarRx videos (Le and Prober

2018)) may be incorporated through a learning

management system (LMS), and 3D images are

more attractive and make students active. The

LMS provides its students with lecture

broadcasts, allowing them to visit a “virtual class-

room”anywhere, anytime, and track student

activities, progress, and compliance (Owolabi

and Bekele 2021). These information

technologies are even more important to complete

the Anatomy E-book curriculum on smartphones,

laptops, or tablets; they are easily accessible

“mobile libraries”that allow students to consult

them even between lectures.

8 Visualization in Anatomy Education 177

8.2.4 Cloud Technology/Educational

Videos

Cloud technology (CT) in medical education

treats students and administrators equally. CT

allows students to access homework anywhere

with internet access, allows teachers to upload

learning materials instantly, and to collaborate

with each other easily, saving money on data

storage. It gives students the opportunity to live

chat and share knowledge on a speciﬁc topic and,

therefore, can be used in a new educational

model. In this technique, students watch the lec-

ture in advance, and this allows for more detailed

and engaging discussions during class. Addition-

ally, group work and analytical activities can take

place in the classroom. The ﬂipped classroom

helps improve learning outcomes, not only in

terms of student performance but also in

internalizing information for better results

(Hurtubise et al. 2015; Hew and Lo 2018).

CT-based tools have been gaining popularity

over the decades, but the pandemic has caused a

paradigm shift in its current acceptability as a

visualization technique in the teaching of anat-

omy. Technologies based on CT are one of

the demanding and actively developing areas of

the world of modern information technology. In

the educational process, the use of CT is becom-

ing more popular and opens up many

opportunities for students, teachers, and educa-

tional institutions. Below are the main domains

of cloud technology in teaching anatomy:

Nowadays, mobile devices dominate the tech-

nology world and have become an essential tool

in learning. Running a full-scale 3D human body

directly from a smartphone or tablet for learning

purposes, hospital practice and even for direct

guidance of medical procedures would become a

trivial method. However, it is still quite tricky due

to software and hardware (Gopalakrishnakone

et al. 2010).

Online Cloud-Based Mobile Enabled 3D

Human Body E-Learning Solution:

This is a platform that can render a complex

3D human body using the power of cloud com-

puting environments. The model uses adaptive

technologies to display dynamic 3D content on

any kind of modern device, from tablets to

smartphones and notebooks. The atlas contains a

detailed and accurate representation of the human

anatomy and an interesting, unique feature: real-

time modiﬁcation of body parts directly related to

an associated disease.

Cloud computing environment: The

functionalities of this E-learning solution is

achieved by using the latest technologies for

advanced browser-based 3D modeling (for a pre-

cise view of the components of the human body)

combined with the outstanding power of

Windows Azure cloud computing services to

ensure prompt and efﬁcient interaction through

the application.

The most important aspect of this technology

is Blob Storage. After exporting and adding the

parts of the body to the app (3D models), each

part is converted into a blob, each containing a

small piece of the human body. This technique

gets beneﬁtted from CDN (Content Delivery Net-

work), which allows the data to be sent from

multiple locations or the closest and most reliable

location to the user.

Since the system is cloud-based, it has “unlim-

ited”processing power. This feature not only

helps to convert the 3D object into a blob and

vice-versa but also does a small pre-processing

phase by converting the classic 3D object to

WebGL (Web Graphics Library) 3D object server

side so that a lot of the processing power is moved

from client side to server side, which allows the

models to be seen on low-performance devices.

Another important aspect that makes this

E-learning unique is the usage of object-oriented

databases. This way, the user can get beneﬁtted

from the elasticity of cloud-based databases to

map all the 3D features (color, size, neighbors,

status, etc.). Mapping can be done not just inside

3D objects themselves but directly into a database

and from there to the app. So, whenever any

editing is done to a 3D object feature, it will

also be stored in the database. They are not

editing the object itself but only changing the

info in the database (Butean et al. 2014).

178 A. Patra et al.

The SaaS (Software as a service) model is

considered one of the biggest advantages of

cloud-based computing. It is common for soft-

ware applications to be available to students for

a low fee or for free, making education accessible

to most students. Textbooks recommended by

universities are often expensive and sometimes

outpace another element of medical education,

including tuition. As a result, many students

refrain from purchasing them. Cloud-based

textbooks are one solution to this problem. Digital

books are usually cheaper; thus, students with

lower incomes can gain access to high-quality

education. The implementation of cloud

technologies removes the ﬁnancial inequalities

between students who may exist in the same

educational environment.

Cloud technologies make learning a simple

and fun experience for participants on both sides

of the learning process. Recently, during the

COVID pandemic, students and teachers have

gained access to this technology for seamless

continuation of anatomy and other curricula and

have greatly appreciated its easy accessibility,

time and money savings, instant feedback, valu-

able information, and inexpensive textbooks.

8.2.5 Virtual Dissection

Over the past decade, the teaching of anatomy has

gradually shifted from wet lab to virtual dissec-

tion, pre-dissected and plasticized specimens, and

virtual 3D body systems. The virtual dissection

table utilizes simulation technology to deliver a

realistic visualization of the 3D anatomical details

of a virtual cadaver. Unlike cadavers, students can

“do-return-repeat”dissection multiple times. The

Synchronized Multiple Visualizer System is soft-

ware for teaching anatomy and physiology,

including certain aspects of histology, compara-

tive anatomy, embryology, and pathology

(Owolabi and Bekele 2021). The virtual dissec-

tion table is a commercial product of Anatomage

(a USA-based company) (Fig. 8.4). This virtual

cadaver table can serve different purposes based

on the student’s needs and the user’s expertise.

These uses may include virtual dissection, virtual

3D atlas, teaching aids, and virtual simulators of

human body anatomy and functions (García et al.

2018).

8.2.6 Radiographic Modalities

The use of visualization technologies in the teach-

ing of anatomy should not be restricted to the

mere demonstration of anatomical images for a

more detailed, interactive, or accessible format

(Bajka 2004). Identiﬁcation of anatomical

structures in clinical images is a professional

requirement for disease diagnosis and treatment.

Thus, the use of imaging in the medical curricu-

lum should also focus on preparing future doctors

to use these technologies appropriately in their

future practice. In this regard, an effective

approach to integrating radiology teaching into

anatomy teaching can be aided by the use of

technology-assisted visualization (Webb 2013).

Computed tomography (CT) and magnetic res-

onance imaging (MRI) are used extensively in the

majority of anatomy courses to understand better

the various clinical conditions discussed in anat-

omy sessions (Mavrych 2016). Recently, medical

technology has provided synthetic cadavers with

organs equipped with an ultrasound probe inside

for better visualization. The use of endoscopy and

colonoscopy to visualize the internal anatomy of

the intestine can make the topic particularly inter-

esting and engaging for students (Patra et al.

2022).

8.2.7 Online or Web Screen

Visualization

Distance online learning and portable networking

devices are becoming an essential component of

the learning environment in anatomy education,

impacting the design and reshaping of the anat-

omy curriculum. Some lectures are delivered

online through systems such as Zoom meetings,

Go - to the webinar, Google classrooms or

Microsoft Teams, etc. These new communication

and information modalities can be used to

improve teaching and learning experiences.

They have the ability to facilitate student learning

and problem-solving potential, allowing for better

integration of the didactic classroom experience

and smoother clinical practice. Technologies can

merge a large amount of factual information in

digitized form to make conventional didactic

lectures more interesting (Mavrych 2016).

8 Visualization in Anatomy Education 179

Fig. 8.4 Anatomy Professor teaching students using virtual dissection table (this is from our medical school, copyright

owned by us and permission obtained from students)

8.2.8 3D Stereoscopy

Stereoscopy refers to increasing or creating the

illusion of depth using binocular vision or a ste-

reoscope. An advanced stereoscope allows

viewers to perceive three-dimensional depth in

two-dimensional images (Holliman 2005). It

was Daniel John Cunningham, a Scottish anato-

mist, who ﬁrst used stereoscopy in the teaching of

anatomy. Stereoscopic 3D instructional videos

are available and affordable via smartphone, a

complementary tool that has proven more beneﬁ-

cial than 2D videos in improving students’

knowledge of anatomical relationships and

reasoning. The most challenging aspect of

learning anatomy is the correlation between the

3D complexity of a structure and its anatomy.

This can be better achieved by using 3D videos

of autopsies or surgeries. The anatomy of the

brain is complicated to learn. As their anatomical

structures are numerous and assembled in a com-

plex three-dimensional (3D) architecture, classi-

cal schematic drawing or two-dimensional

(2D) photography has proven difﬁcult in

providing a simple, clear, and accurate message.

Advances in photography and computer science

led to the development of stereoscopic 3D visual-

ization, ﬁrst for entertainment and then for educa-

tion. Stereoscopic 3D teaching of neuroanatomy

has sparked enthusiasm for digital technology

among medical students. It could improve their

anatomical knowledge and test results, as well as

their clinical competence. Depending on the

institute’s possibilities and the teachers’commit-

ment, this new modality should also be extended

to other anatomical disciplines. However, its

implantation requires trained faculties and its

impact on clinical competence needs to be objec-

tively assessed (Jacquesson et al. 2020).

Incorporating 3D videos as supplementary teach-

ing in the curriculum could enhance students’

knowledge of anatomical relationships and

reasoning. (Bernard et al. 2020).

180 A. Patra et al.

8.2.9 Interactive 3D Computer

Graphics (3DCG) Model

Medical students frequently ﬁnd it difﬁcult to

conceptualize 3D anatomical structures, such as

bone alignment, spatial muscle anatomy, and

complex movements, from 2D images (Battulga

et al. 2012). Although cadaveric dissection allows

haptic understanding of 3D anatomical structures,

it is expensive and time-consuming (McLachlan

and Patten 2006). In addition, the time allocated

to complete the anatomy curriculum decreases

year by year (Cottam 1999; Drake et al. 2009),

leading to increased cognitive load and hindering

the learning of anatomy for students with poor

spatial skills (Garg et al. 2001; McLachlan et al.

2004; Levinson et al. 2007; Huk 2006). Thus, the

current trend is to achieve anatomical understand-

ing in a short period by incorporating innovative

educational technologies in anatomical education.

Animated and interactive three-dimensional com-

puter graphics (3DCG) models are a relatively

new technology that can approximate human

anatomy and movement. 3DCG models have

shown promise in achieving a spatial understand-

ing of 3D anatomy from 2D images (Fig. 8.5) and

text and in overcoming many of these learning

tasks. According to Mayer’s cognitive theory of

multimedia learning, students learn best with both

words and pictures in an electronic learning envi-

ronment (Mayer 2009). 3DCG visually conveys

semi-real information to students, allowing them

to easily grasp the content. In addition, the inter-

active 3DCG content also improves students’

cognitive abilities.

Several institutions have developed 3DCG

animation and interactive 3DCG (Silén et al.

2008). Kobayashi et al. reported that it was

much easier and more accurate to explain the

details of surgical procedures using 3DCG ani-

mation than 2D illustration (Kobayashi et al.

2006). In addition, methods have been developed

to allow users to create highly specialized 3DCG

content on the web. The use of 3DCG models has

beneﬁts over traditional methods of teaching

anatomy; however, their development and adop-

tion are time-consuming. Therefore, new educa-

tional methods of teaching information and

communication technologies are needed.

8.3 Recent Advances

in Visualization

8.3.1 Anatomy Studio

3D reconstruction from anatomical sections

allows anatomists to develop 3D representations

of real anatomical structures by tracing organs

from cryosection sequences. However, conven-

tional user interfaces depend on a single user

experience to develop content for educational or

training purposes. Anatomy Studio, a mixed real-

ity (MR) tool for virtual dissection through aug-

mented three-dimensional reconstruction (3DR),

combines tablet drawing with touch and transpar-

ent head-mounted displays with MR-based visu-

alization to perform 3DR of anatomical

structures. This is a new interactive technique

designed to support spatial understanding and

speed up manual segmentation. Using in-air

interactions and interactive surfaces, instructors

can easily access the cryosection and edit

contours while watching other users’

contributions. A user study involving experienced

instructors and medical professionals conducted

in actual working sessions shows that Anatomy

Studio is suitable and beneﬁcial for 3D recon-

struction. The results suggest that Anatomy Stu-

dio supports close-knit collaboration and group

discussion to gain deeper insights. 3D visualiza-

tion provides enhanced perceptual interaction

with Anatomy Studio using in-air gesture collab-

oration that shows real-time contour changes

made by others, thus facilitating communication.

The anatomical study includes a drawing table,

and the user is equipped with a tablet, a stylus,

and a transparent display mounted on the head.

Using a hand gesture, the user can build a 3D

virtual model on the MR (mixed reality) dissec-

tion table (Zorzal et al. 2019).

8 Visualization in Anatomy Education 181

Fig. 8.5 Description of

structure and outcome with

local coordination

(assembled 3DCG

animated model from 2D

illustrations)

8.3.2 Artificial

Intelligence/Humanoid Robots

Artiﬁcial intelligence (AI) refers to the capacity of

a computer/robot to perform human-like tasks

with respect to cognition (Russell and Norvig

2003). An intelligent system has the best chance

of achieving this goal, as it is aware of its sur-

roundings and can act appropriately. This was

aptly called as “AI,”to describe automated

computers that imitate human “cognitive”

activities like “learning”and “problem-solving.”

Our daily lives are surrounded and inﬂuenced by

a wide range of AI applications, including self-

driving cars to humanoid robots, human speech

comprehension applications (such as Siri and

Alexa), suggestion tools (such as Vimeo,

Facebook, and iTunes), sophisticated electronic

databases (such as Google), and suggestion tools

(Poole et al. 1998; Asghar et al. 2022). In recent

days, the use of Robots to perform different

surgeries is becoming popular. Humanoid robots

have recently been used as caregivers in many

hospitals to protect doctors and nurses from the

pandemic. Although many patient care services

can be handled by robotics, including screening,

disinfection, surgery, telehealth, and logistics,

their role in medical education and academia is

unclear (Shen et al. 2021; Asghar et al. 2022).

Exempliﬁcation and the capacity to integrate

social interaction into the learning environment

are fundamental qualities of educational robots

that guide in viable learning among students.

Signiﬁcant progress has been made in the creation

of such a robot with a musculoskeletal system that

is comparable to that of a human. Aldebaran

Robotics created the autonomous, programmable

humanoid robot NAO for use in educational and

research settings to map humanoid movement

onto a reinforcement learning task (García and

Shaﬁe2020; Asghar et al. 2022) These robots

are more beneﬁcial than computer software or

other educational aids in many instructional

goals because they can mimic human responses

(Tuna and Tuna 2019). They are so well-

equipped with cutting-edge information and orig-

inal teaching methods. Humanoid robots can also

be trained to know exactly what each student

needs to know, which could help with customized

instruction. In light of the numerous possibilities

that humanoid robots can provide, we have

attempted to investigate their current use in edu-

cation, their potential roles and functions in the

instructional delivery of Anatomy, and the

challenges in this domain.

182 A. Patra et al.

The Role of a Robotic Assistant in the Era

of Virtual Anatomy

The conventional, century-old method of teach-

ing and learning anatomy is human cadaveric

dissection. If the organ is too small to dissect or

difﬁcult to visualize, like the pituitary, prostate,

and adrenal glands, it is difﬁcult for the anatomist

to reach it. While robotic dissection with

expanded vision has enabled the display of intri-

cate anatomical structures. According to Dal

Moro (2018), a robotic dissection is a team

approach that involves a robotic assistant at the

corpse and an anatomist at the console. With a

magniﬁed three-dimensional (3D) view and more

advanced wrist-hand movements than human

assistance, a robotic assistant might, for instance,

handle delicate tissue. Using 3D eyeglasses, a 3D

view of the dissected can be seen as a byproduct

of robotic dissection and with the assistance of a

large 3D monitor (Zhang et al. 2019).

Additionally, during the dissection, the clinical

or surgical anatomy could be explained. As a

result, residents and medical students could

beneﬁt greatly from robotic dissection as a teach-

ing tool. Hence robotic dissection could be an

effective education program that can be devel-

oped and tested. A computer-simulated environ-

ment is provided by virtual reality simulators to

enhance one’s ability to dissect. Computer

connections are necessary for both tactile dissec-

tion and virtual reality simulators; Consequently,

they will continue to grow simultaneously.

Students will undoubtedly be able to develop

their surgical skills right from the start of their

medical training if cadaveric dissection is

performed using surgical robots.

8.3.3 High Fidelity Simulation

High Fidelity Simulation uses sophisticated

mannequins in simulated patient environments.

It is also called a human patient simulator or

high-ﬁdelity simulator. This method uses

computers to control full-body mannequins that

are programmed to provide realistic physiological

responses to student actions. Normally, these

mannequins can demonstrate physiological and

pathophysiological processes and respond to

interventions, making them very useful for teach-

ing complex phenomena and integrating multiple

dimensions of basic medical science into individ-

ual sessions. For example, mannequins can sleep

and wake up, breathe and talk, and are also capa-

ble of giving birth, bleeding, and vomiting. They

can be utilized to learn and manipulate almost all

signiﬁcant vital and physiological signs such as

pulse, heart rate, EKG, and more. All these

features allowed students to better present

structures, functions, symptoms, and medical

skills (Lewis et al. 2012).

8.3.4 Immersion or Haptic

Technology

This technology refers to tactile-based feedback

technology, where the sense of touch is applied

by applying motion to the user’sﬁngertips. Vir-

tual Reality (VR), Augmented Reality (AR), as

well as other recent technologies provide a unique

scenario (Motaharifar et al. 2021). AR has a vari-

ety of approaches for teaching health

professionals of all ages. Students of different

levels can beneﬁt from computer-generated

simulations. With Human–Computer Interface

(HCI), students can engage and experience virtual

environments. They differ in terms of ﬁdelity,

immersion, and interactivity. Immersive high-

deﬁnition visual inputs create accurate digital

representations of the real anatomical structures

in virtual reality. Head-mounted displays

(VR headsets), motion sensors, controllers,

keyboards, and speech recognition software are

used to interact with the virtual world. In contrast,

AR superimposes computer-generated stimuli on

real-world environments/objects, such as

computer-generated anatomical structures

superimposed on a mannequin (Othmana and

Amiruddinb 2010). Alternate reality platforms

provide an alternate world where users can

engage with the story and inﬂuence it by making

choices. Participants interact with the virtual

environment using real-world technology in alter-

nate reality simulations, like interfacing with

patient data using electronic health record

simulations. Although the forms exist on an

overlapping continuum often characterized as

“mixed reality,”the degree of immersion in the

virtual environment distinguishes VR from AR

and alternate reality (Fig. 8.6).

8 Visualization in Anatomy Education 183

Fig. 8.6 Recent advances in visualization and its possible uses

8.4 Further Thoughts

and Recommendations

8.4.1 Merits and Demerits of Using

3D Visualizations

Undoubtedly, the visualization of anatomy and

related basic medical subjects could be improved

through strategic adjustments, media, and tech-

nology (Owolabi and Bekele 2021). The pan-

demic provided insight into the advantages and

disadvantages of these technologies and

innovations. The continued integration of tech-

nology into anatomy education is certainly neces-

sary to improve learning outcomes and address

the cognitive load related to the volume of anat-

omy education and training (Gaur et al. 2020).

Traditional teaching practice such as cadaver dis-

section is almost synonymous with teaching anat-

omy. However, if the lack of corpses continues, it

would be impractical to stop or continue teaching

anatomy with a limited number of cadavers.

Thus, technology remains a reliable way to ensure

that students do not suffer from deﬁciencies.

Therefore, judicious use of technology and crea-

tive adaptations is necessary to ensure the timely,

efﬁcient and effective provision of anatomical

education despite the limitations that have devel-

oped during or even after the pandemic (Saverino

2020). For example, it is also evident in the

United Kingdom that such adjustments are being

made with evidence of success (Longhurst et al.

2020).

184 A. Patra et al.

8.4.2 Limitations

It is essential to note that despite the enormous

advantages presented by the adoption of technol-

ogy that has empowered constant realization,

there have been various constraints for the same.

These restrictions incorporate unfeasible visual

techniques, discriminatory access, cost viability,

and absence of preparation among anatomy

educators. The objective of all educational

innovation or technology is to develop learning

further, yet we have shown that adding dynamic

simulation affects the people who likely need

it most: students with somewhat poor spatial

capacity. Bodies provide real-time learning for

medical students, while virtual cadavers provide

a realistic visualization of 3D anatomical detail.

Post-mortem training or procedure imparts in

youthful personalities the qualities of empathy,

professionalism, ethics, and conﬁdentiality.

Learning anatomy through virtual dissection is

unrealistic (Basavanna et al. 2022). Accordingly,

virtual modalities can be utilized as an adjunct to

envision the anatomy of complicated structures

(middle ear cavity or ethmoid cavity).

8.5 Concluding

Statements/Take-Home

Message

Anatomy education and teaching medicine as a

profession was never designed to be fully virtual.

However, the use of technology-based online

pedagogies (3D learning management system,

anatomy studio, virtual classroom, medical

simulations, virtual cadavers, 3D stereoscopy,

etc.) has been proven to play a pivotal role in

the successful completion of the Anatomy curric-

ulum. As discussed, there are a plethora of

technologies that aid in learning anatomy.

Policymakers need to think critically about how

to amalgamate medical informatics and conven-

tional pedagogical approach to timely reform

anatomy education. Interactive 3D visualization

technologies have undoubtedly made the subject

of Anatomy extremely visual. Consequently,

anatomy, conventionally a teacher-centric sub-

ject, is gradually becoming learner-centric with

the use of readily available online pedagogies,

and rightly so. There is no harm in using readily

available study materials available online, but the

content available should not be trusted blindly but

rather be cross-checked; this is the role of con-

ventional textbooks and teachers. Last but not the

least, we should always remember that the ﬁnest

way to teach and learn modern Anatomy is by

blending conventional pedagogy with multi-

modal system-based approaches to complement

one another.

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Visualizing Anatomy in Dental

Morphology Education 9

Tamara Vagg, Andre Toulouse, Conor O’Mahony,

and Mutahira Lone

Abstract

Tooth morphology is a foundation course for

all dental healthcare students including

dentists, dental hygiene, dental therapy, and

dental nursing students. This chapter explores

the conventional and innovative teaching

methods to deliver tooth morphology educa-

tional modules. The teaching tools are

explored with a 2D and 3D lens, with a partic-

ular focus on visualization, student under-

standing, and engagement. Traditional

methods of teaching tooth morphology must

be complemented with innovative pedagogical

approaches in order to maintain student’s

attention and accommodate their diverse

learning methods. Teaching 3D anatomy

enables students to visualize and spatially

comprehend the link between various

anatomical components. Online tests and quiz-

zes motivate students and are also beneﬁcial in

preparing students for exams. Online self-

examinations offering visualization with 3D

teeth enable students to evaluate their knowl-

edge and offers immediate feedback, which

aids in the long-term retention of information.

These tools can be as efﬁcient as other teach-

ing methods, allowing the students to study at

their own pace and with repetition. The

authors conclude that blended and innovative

teaching methods should supplement student

learning and not replace, traditional face-to-

face educational methods.

T. Vagg

Cork Adult CF Centre, Cork University Hospital,

University College Cork, Wilton, Cork, Republic of

Ireland

School of Computer Science and Information Technology,

University College Cork, Cork, Republic of Ireland

HRB Clinical Research Facility Cork, University College

Cork, Cork, Republic of Ireland

e-mail: tamara.vagg@ucc.ie

A. Toulouse · C. O’Mahony · M. Lone (✉)

Department of Anatomy and Neuroscience, University

College Cork, Cork, Republic of Ireland

e-mail: A.Toulouse@ucc.ie;conoromahony@ucc.ie;m.

lone@ucc.ie

#The Author(s), under exclusive license to Springer Nature Switzerland AG 2023

E. Abdel Meguid et al. (eds.), Biomedical Visualisation, Advances in Experimental Medicine and

Biology 1406, https://doi.org/10.1007/978-3-031-26462-7_9

187

Keywords

Digital teaching · Digital divide · Tooth

morphology · Dental anatomy · 3D

9.1 Background

Anatomy is a core module that provides a strong

foundation in all healthcare programs including

the undergraduate and postgraduate dental curric-

ulum (Sugand et al. 2010; Abdalla 2020;

McHanwell and Matthan 2020). Prior to patient

examination, diagnosis, and clinical treatment, a

thorough grasp of anatomy is required (Abdel

Meguid et al. 2017; McHanwell and Matthan

2020; Reddy and Pathak 2021; Fonseca et al.

2022). Vesalius’traditional method of

teaching anatomy via dissection has evolved with

an emphasis on didactic lectures but now also

includes digital education, e-learning, and a

broad variety of 3D models (Nagasawa et al.

2010; Salajan et al. 2015; Lone et al. 2018)a

the current generation of students are increasingly

using devices like computers/laptops and cell

phones for their academic needs (Khatoon et al.

2014). Additionally, visualization has been found

to be particularly vital for student comprehension

as well as the development of psychomotor

abilities, which are particularly important for a

dental student (Fonseca et al. 2022).

188 T. Vagg et al.

The term “dental student”has evolved over

time to include not just those pursuing a dental

science degree (bachelor’s or doctorate,

depending on the region), but also those pursuing

degrees in dental hygiene, dental therapy, or den-

tal nursing (Bakr et al. 2016; McHanwell and

Matthan 2020). As they begin their respective

degree, these students will treat patients by

performing oral examinations, dental scaling,

restorations, and small surgeries; thus, in order

to promote safe clinical practice, anatomical

knowledge is a crucial part of their early training

(McHanwell and Matthan 2020). General gross

anatomy, embryology, neuroanatomy, histology,

and oral anatomy (including tooth morphology)

are among the anatomical topics that are typically

covered in the ﬁrst 2 years of a dental study

program (McHanwell et al. 2007; Gould et al.

2014; McHanwell and Matthan 2020; Chow and

Sharmin 2021), with a core anatomy curriculum

published by the Association for Dental Educa-

tion in Europe (ADEE) (ADEE 2016; Best et al.

2016). Dental students need to cover certain

anatomical topics in more detail than other

healthcare students including the anatomical

structures of the oral cavity, major and minor

salivary glands, maxilla, mandible, nerve supply

to the teeth, ﬂoor of the oral cavity, and the

lymphatic drainage of the head and neck to

name a few (Guttmann et al. 2003; ADEE

2016). There is a shortage of qualiﬁed dental

anatomy staff available to teach anatomy to dental

students, especially those who can emphasize the

dental relevance of the anatomical structures

(McHanwell 2015). Furthermore, horizontal and

vertical integration of anatomy into the dental

curriculum is recommended, which is extremely

relevant in the clinical years (Manogue et al.

2011; McHanwell and Matthan 2020).

Courses in dental anatomy are normally taught

as either separate modules or as an integrated

anatomy module (Guttmann et al. 2003).

Co-teaching, in which students of different dental

specialties learn alongside one another, is another

strategy that has been proposed as a means of

enhancing students’capacities in the areas of

communication and teamwork in preparation for

the students’future roles as members of the dental

care team (Bakr et al. 2016; Amin et al. 2017;

McHanwell and Matthan 2020).

In the following sections of this chapter, the

conventional and innovative teaching tools used

for tooth morphology will be discussed with a

focus on instructional tools employed with visu-

alization at its core.

9.2 Tooth Morphology

Tooth morphology is the study of the morpholog-

ical features of the permanent and deciduous

dentitions (Obrez et al. 2011). Dental students

including dental hygienists, dental nurses, and

dental technicians must be familiar with tooth

morphology including the visualization and

understanding the 3D aspects of different teeth

for clinical and lab work (Obrez et al. 2011; Abu

Eid et al. 2013; Bakr et al. 2016; Risnes et al.

2019). The tooth morphology module is usually

delivered in the ﬁrst 2 years of the dental degree

(Lone et al. 2018) with its application in the

subsequent years. This often leads to a loss of

knowledge called “decontextualized technique

learning”(Magne 2015). The teaching of tooth

morphology has always been an important but

difﬁcult aspect (Wang et al. 2020) with various

teaching aids available to assist student learning

and understanding. Tooth anatomy and identiﬁca-

tion is not only important for the clinical treat-

ment and restoration of teeth but also for the

identiﬁcation of dental anomalies, dental

variations, and pathologies commonly seen in

clinical practice (Bakr et al. 2016). In the next

section, the authors will discuss the visualization

offered by conventional (2D and 3D) methods for

teaching tooth morphology.

9 Visualizing Anatomy in Dental Morphology Education 189

Fig. 9.1 Timeline showing the 5-year curriculum at University College Cork (UCC). Tooth morphology is taught in

years 1 and 2 with vertical integration needed in the following years (years 3–5)

9.3 Conventional Methods

of Teaching Tooth Morphology

Which Offer Visualization

Modalities used for teaching tooth morphology

include a variety of approaches, such as lectures

supplemented with prosected material,

anatomical models, and extracted teeth (Lone

et al. 2018) (Fig. 9.1). As this chapter focuses

on visualization offered by each teaching method,

the teaching modalities are further subdivided

into 2D and 3D teaching methods.

9.4 2D Teaching Methods

9.4.1 Lectures

Didactic lectures utilizing PowerPoint (Shigli

et al. 2016) are often the preferred medium for

teaching dental anatomy (Johnson et al. 2012;

Schonwetter et al. 2016; Lone et al. 2018). Dental

anatomy is a topic that easily lends itself to being

taught in this style since it is so readily supported

by visual media. Furthermore, viewing multiple

two-dimensional representations of the same

structure may help to create a 3D mental image.

Anatomy didactic lectures can be supplemented

with other teaching modalities to offer multiple

entry points and provide multimodal and holistic

teaching, as will be discussed in the following

section; however, when used on its own, this

mode of instruction can assist students in devel-

oping mental images of the structures.

During the recent pandemic, online lectures

and webinars substituted the didactic lecture con-

tent in dental schools globally (Goodacre et al.

2021; Lone et al. 2021). As we return back to

face-to-face teaching, the lecture

recordings would be a helpful student resouce as

a review tool to explain certain points for visual

learners as well as for auditory learners.

According to Bacro et al. (2013), there is substan-

tial association between grades for students with a

preference for auditory learning and frequent

viewings of lecture recordings. The authors

hypothesize that auditory learners would

greatly beneﬁt from having access to recorded

lectures (Bacro et al. 2013).

190 T. Vagg et al.

9.4.2 Flash Cards

Flash cards offer a 2D representation of tooth

anatomy which aids visualization and allows for

multiple viewings according to user suitability.

These ﬂash cards are readily available in

textbooks (Nelson 2014; Wignall 2014;

Fehrenbach 2015), provide a detailed description

of the features of the teeth, promote student

learning, and facilitate a transfer of knowledge

from 2D to 3D (Bryson 2012). Flash cards can

also be offered to dental students as case-based

studies in the clinical years (Al-Rawi et al. 2015;

McAndrew et al. 2016; Toyohiro et al. 2021).

9.5 3D Teaching Methods

9.5.1 Dissection- and

Prosection-Based Teaching

An ongoing debate exists about whether dissec-

tion or prosection-based practical sessions are the

superior approaches for teaching anatomy. Both

dissection sessions and prosections give visual

cues and aid dental students in gaining a better

understanding of the head and neck region, par-

ticularly the teeth in occlusion and their relation-

ship to the surrounding anatomical structures in

the oral cavity. Prosection-based instruction is

often favored by students themselves (Abdel

Meguid et al. 2017). Snelling et al. (2003)

conducted a poll of dental students and the

students rated tutorials, prosections, and

textbooks as excellent anatomy learning tools

(Snelling et al. 2003).

Redwood and Townsend (2011) state that den-

tal students’interest in pursuing a dental surgical

career may explain why they choose prosection

over dissection as the preferred learning method.

Regardless of the reasons stated above,

prosections and dissection sessions should remain

a core teaching component of the dental curricu-

lum since it aids student development into

clinicians, with the cadaver as their ﬁrst teaching

patient, and also provides 3D understanding and

visualization of the anatomical relationship of the

teeth and other anatomical structures in the oral

cavity (Redwood and Townsend 2011).

9.5.2 Anatomical Models

Teaching tooth morphology commonly employs

the use of plastic or resin-based anatomical

models (Lone et al. 2018). The models are easily

available, convenient to use and store and have a

long shelf life (Abu Eid et al. 2013). Students,

themselves, appreciate the 3D teaching offered by

plastic models and use it routinely to supplement

their learning (Abu Eid et al. 2013; Abdel Meguid

et al. 2017).

Tooth morphology taught with the use of high-

quality commercial replicas with color-coding or

numbering of certain tooth characteristics has

visually emphasized the differentiation of tooth

characteristics, further aiding student understand-

ing (Abu Eid et al. 2013). However, one disad-

vantage is that all the plastic teeth are identical

and do not show normal anatomical variations or

anomalies (Obrez et al. 2011).

9.5.3 Extracted/Plastic Teeth

Historically, extracted teeth are the most popular

teaching aid for studying dental morphology and

identifying tooth characteristics (Mitov et al.

2010). According to research by Abu Eid et al.

(2013), students prefer to study dental morphol-

ogy using extracted teeth (Abu Eid et al. 2013).

However, there are various drawbacks for teach-

ing with extracted teeth including obtaining a

sufﬁcient number of hygienic and healthy,

non-carious, unworn extracted teeth with ethical

and informed consent (Cantín et al. 2015).

In most countries, improvements in oral health

care have led to a decline in the number of tooth

extractions (Muller et al. 2007; Dietrich et al.

2015), posing a further challenge for the procure-

ment of healthy extracted teeth for educational

purposes (Obrez et al. 2011). Innovative teaching

methods are currently being incorporated into the

teaching of tooth morphology (Nagasawa et al.

2010; Cantín et al. 2015; Lone et al. 2021)i

order to provide visualization and 3D comprehen-

sion of the dental morphological anatomy (Mitov

et al. 2010; Allen et al. 2015). However, extracted

teeth are still preferred for learning tooth mor-

phology and also for examinations (Suh et al.

2022).

9 Visualizing Anatomy in Dental Morphology Education 191

9.5.4 Carving Teeth

Carving tooth models from various media, such

as wax, chalk, or soap (Mitov et al. 2010; Lone

et al. 2018), has helped facilitate the learning of

comprehensive dental morphology (Obrez et al.

2011). This method utilizes visualization and

haptic touch to provide the development of ﬁne

motor skills along with morphological knowl-

edge, which is a prerequisite for reconstructing

lost or damaged tooth structure during various

dental treatments (Abu Eid et al. 2013;La

et al. 2015; Goodacre et al. 2021). A study

done in 2013 introduced wax tooth carving

practical sessions for ﬁrst-year graduate

dentistry students with more than 80% of the

students claiming that the carving assignments

improved their manual dexterity along with

their understanding and ability to visualize

the 3D architecture of the teeth (Abu Eid et al.

2013).

More recently, Abdalla (2020) supplemented

dental carving exercises with clinical-based

teaching and also digital learning to improve stu-

dent satisfaction and student performance.

Introducing clinical scenarios allowed students

to see the relevance and application of tooth anat-

omy in their clinical practice and careers. The

digital teaching introduced for the tooth morphol-

ogy module linked directly to the digital software

used in dental practices and hence allowed the

dental students to link their pre-clinical haptic

exercises to digital uses and clinical applications

(Abdalla 2020). At-home waxing exercises

(Goodacre et al. 2021) and 3D tooth models

(Lone et al. 2021) were also found to be effective

for teaching students the didactic aspects of vari-

ous teeth during the recent COVID-19 pandemic.

However, the students expressed an interest in

returning to campus for these tooth morphology

modules.

There appears to be a debate about the beneﬁts

of dental carving exercises and whether they

should be retained or introduced/reintroduced in

the tooth morphology module (Conte et al. 2021).

Goodacre et al. (2021) favors the carving sessions

for the students as it not only enhances

their detailed tooth morphology knowledge but

also introduces 3D spatial understanding of the

tooth along with detailed hand manipulation of

dental instruments, which will all be relevant

skills needed in the dental care of the patients

(Goodacre et al. 2021).

While the sections above discuss the various

teaching methods, self-assessment with mock

quizzes or spots can also be used to enhance

student learning and motivate them. The next

section discusses how this has been implemented

for tooth morphology teaching.

9.5.5 Formative “Spotter”

Examination

Self-assessment tools have been found to enhance

student learning with better results and motiva-

tion (Lone et al. 2019). Tooth morphology,

computer-based learning program for tooth mor-

phology showed promising results with improved

student learning (Bogacki et al. 2004). The major-

ity of dental schools evaluate tooth morphology

formally as part of an objective structured practi-

cal examination or formative spot examination

which consists of stations where students are

asked to identify extracted or plastic teeth and

chart them in an appropriate notation system and

answer a clinically or developmentally related

question (Abu Eid et al. 2013; Lone et al. 2019).

9.5.6 Innovative Visual Methods

for Teaching Tooth Morphology

to Dental Students

Dental students require detailed knowledge of the

morphology of the permanent and deciduous

teeth. Technology has been used successfully in

pre-clinical and clinical dental teaching (Suh et al.

2022). The challenge for educators is to provide a

framework so that students are offered a learning

environment that includes all different types

of learners. Providing the students the opportu-

nity to learn both online and in-person, gives all

the learners a chance to get a better understanding

of the best way for them to learn. In the last few

years changes in the anatomy curriculum along

with the recent COVID-19 pandemic (Drake and

Pawlina 2014; McBride and Drake 2018;

Longhurst et al. 2020; Dulohery et al. 2021)

resulted in reduced course hours for anatomy.

This led to an increase in the use of adjunctive

tools and self-directed learning which have been

demonstrated to be efﬁcient as teaching resources

while also being easily accessible for students

(Tam et al. 2009; Yeung et al. 2011; Goodacre

et al. 2021; Lone et al. 2021). Medical students

are routinely utilizing newly created and tested

instructional strategies (Rizzolo and Stewart

2006; Johnson et al. 2012) which have also been

incorporated into the dental curriculum (Maggio

et al. 2012). In addition to this, a survey

conducted by Lone et al. (2018) investigating

the tooth morphology teaching methods

employed within the United Kingdom and

Ireland, found that minimum time is dedicated

to the delivery of tooth morphology resulting in

a greater emphasis on students’self-directed

learning. The authors also conclude that Com-

puter Aided Learning (CAL) systems and tools

could assist in acquiring and sustaining tooth

morphology learning (Lone et al. 2018). Further-

more, since dental students use the latest elec-

tronic learning devices, these CAL systems and

online tools for self-directed dental education are

easily accessible (Redwood and Townsend

2011). Indeed, a study conducted at the School

of Dentistry, University of Birmingham found

that dental students preferred devices like laptops

and smartphones for studying and self-testing

their knowledge (Khatoon et al. 2014). What

follows is a review of the current literature,

exploring the use of innovative tools offering

visualization to teach tooth morphology to dental

students.

192 T. Vagg et al.

9.5.7 Animations and Multimedia

Learning Resources

The use of graphics and visual information

enhances understanding, retention, and recall of

a theory or notion (Tversky et al. 2002). Teaching

modalities using both static images (such as

pictures and diagrams) and dynamic images

(such as videos and animations) are essential in

stimulating and creating an enhanced environ-

ment for teaching and learning (Wilson 2015).

Animations serve two functions in an educational

context: affective and cognitive. The affective

component of an animation keeps the viewers’

attention, while also working as a motivational

tool. On the other hand, the cognitive component

of animation is associated with comprehension

(Lowe 2004). Regardless of the results obtained,

students have expressed enthusiasm toward the

use of technology-based courses or modules

within the curriculum (Kesner and Linzey 2005;

Vuchkova et al. 2012).

Animation and various multimedia learning

tools have been developed for dental students to

teach topics like cranial nerves (Richardson-

Hatcher et al. 2014; Lone et al. 2017) (Fig. 9.2),

temporomandibular joint and dental anesthesia

(Guttmann 2000) and the 3D morphology of the

teeth (Lone et al. 2019). According to Mitov et al.

(2010), their 3D animated tooth software proved

effective and enjoyable for learning tooth mor-

phology (Mitov et al. 2010).

Bogacki et al. (2004) conducted a randomized

controlled trial assessing the efﬁcacy of “Tooth

Morphology,”an interactive CAL course with

lectures containing text, illustrations and 3D

graphics, against traditional lectures for teaching

tooth morphology (Bogacki et al. 2004). For a

cohort of ﬁrst-year dental students at Virginia

Commonwealth University, the results found

that “Tooth Morphology”was statistically equal

to traditional teaching lectures. These discoveries

have resulted in the substitution of conventional

lectures with a mix of “Tooth Morphology”and

interactive classroom discussions since this new

format was deemed to be more interactive and

gave students active control over the time and

pace of their learning. It also increased chances

for teachers to connect with the students, provide

assistance and emphasize the clinical importance

of tooth morphology (Bogacki et al. 2004).

9 Visualizing Anatomy in Dental Morphology Education 193

Fig. 9.2 Screenshot of the cranial nerve animation showing the 12 pairs of cranial nerves with individual functions.

Sensory supply is demonstrated in green whereas motor supply is shown in orange

Students’use of a DVD-based interactive

tooth atlas was studied by Wright and Hendricson

(2010). The atlas included photographic, radio-

graphic, and CT scan images of all the teeth and

was offered to the dental students in the ﬁrst three

years of their degrees at the University of Texas

Health Center, San Antonio. Before performing

surgical procedures like periapical and implant

surgeries these atlases could be reviewed to ana-

lyze the anatomy of various teeth and their rela-

tionship to the surrounding hard and soft tissues.

Only 14% of dental students downloaded the

atlas. However, once students were informed

that atlas-based questions would be featured on

the exam the uptake of the atlas increased to 43%

of students (Wright and Hendricson 2010). In

addition, the authors of the study determined

that the atlas was mostly used by third-year dental

students, presumably because they were involved

with the clinical rotations and could comprehend

the clinical signiﬁcance of the anatomical infor-

mation available in the resource (Wright and

Hendricson 2010).

194 T. Vagg et al.

In the aforementioned examples, the

animations and multimedia learning resources

were limited in terms of their interaction

capabilities and accessibility to students, which

is reﬂective of Information Communication

Technologies (ICT) for that time. However,

since then, innovative visualization methods

have continued to advance, with the emergence

of a new cycle of interactive 3D programs capable

of virtual/augmented/mixed realities and even

delivery through web platforms. The proceeding

sub-sections will explore how these innovations

have been utilized for dental/tooth morphology.

9.5.8 3D Models, 3D Animations

and Interactive 3D

There are numerous beneﬁts for employing novel

teaching tools, such as simultaneous visualization

of material by multiple students and the applica-

tion of teaching and clinical training concurrently

(Nagasawa et al. 2010). Continuous

improvements in technology over the years have

led to the development of 3D teaching tools for

dental students such as interactive 3D tooth

(de Boer et al. 2015) or 3D tooth atlases (Salajan

and Mount 2008; Nagasawa et al. 2010; Wright

and Hendricson 2010; Salajan et al. 2015; Suh

et al. 2022), including the development of an tooth

atlas using micro CT scanning (Nagasawa et al.

2010; Cantín et al. 2015; Koopaie and Kolahdouz

2016), and 3D visualization of the internal tooth

structure (Salajan et al. 2015). Atlases have been

provided to students via CD/DVD (Wright and

Hendricson 2010) or more recently, via

web-based or online delivery (Salajan et al.

2015). Also, the use of other 3D delivery

solutions/software such as Computer-Aided

Design (CAD) and Computer-Aided

Manufacturing (CAM) technology aided student

performance in wax-up and restoration of teeth

(Douglas et al. 2014). Furthermore, tooth

morphology may be taught in a safe and cost-

effective manner using 3D scanning of extracted

teeth (Elgreatly and Mahrous 2020).

Mitov et al. (2010) introduced a 3D tool via

web-based CAL software “MorphoDent”to den-

tal students in their second year of study at the

University of Saarland in Homburg, Germany.

Scanners were utilized to build 3D models of

extracted teeth which were previously used for

the teaching tooth morphology. Students were

provided with the teaching tool two weeks before

examination in order to evaluate their perception

about the efﬁcacy of the 3D teaching tool. 3D

tooth models were also examined, in addition to

the standard examination using extracted teeth.

Data gathered shows that while students liked

learning with MorphoDent, no signiﬁcant statisti-

cal difference was seen between the outcome of

the two examination scores (Mitov et al. 2010).

However, Lone et al. (2018) developed an inter-

active 3D tooth morphology quiz and evaluated

its effects on dental education via a cross-over

study designed to compare the novel system

with traditional teaching methods. Students who

used the 3D tooth morphology quiz felt the sys-

tem was intuitive and aided in their understanding

of tooth morphology. The results of the study

found that student groups with access to the 3D

tooth morphology quiz performed signiﬁcantly

better than previous years. The authors conclude

that the 3D tooth morphology quiz is a useful

adjunct for dental anatomical education (Lone

et al. 2019) (Fig. 9.3).

Magne (2015) revised and implemented a sig-

niﬁcant update of the dental morphology and

occlusion module at Herman Ostrow School of

Dentistry in the University of Southern California

by introducing 2D, 3D, and 4D principles for

learning essential practical and clinical skills.

Initially, his method focused on 2D tooth

drawings, proceeded by partial or full 3D

wax-ups of teeth. 4D was utilized by layering

acrylic and resin restorations simulating the dif-

ferent layers of the internal structure of the tooth,

namely enamel, and dentine. Increased student

engagement along with staff satisfaction was

reported with the modiﬁed program (Magne

2015).

9 Visualizing Anatomy in Dental Morphology Education 195

Fig. 9.3 Tooth morphology quiz (TMQ). (a) Active quiz

screen showing Fédération Dentaire Internationale (FDI)

notation placements of the teeth along with a 3D rotating

image of selected tooth. (b) Results of the TMQ with

feedback screen. Selecting an incorrect answer will high-

light it in yellow with relevant feedback on identiﬁcation

features provided

Extracted teeth, as discussed before, are rou-

tinely used for studying the morphology of the

teeth. Game-based learning and gamiﬁcation has

also been applied for the teaching of tooth

morphology using extracted teeth, with results

showing it to be cost-effective, student-friendly,

and providing 3D understanding for all

the learners (Risnes et al. 2019).

196 T. Vagg et al.

As part of a dental anatomy module redesign at

the University of Kentucky College of Dentistry,

Abdalla (2020) incorporated a 3D dental software

program (“Tooth Explorer”), designed to teach

oral anatomy and tooth morphology. The soft-

ware was employed during lectures on a second

screen where a 3D view of the tooth was

displayed, illustrating anatomical landmarks and

unique features. The authors report that the

redesigned module as a whole, including the use

of “Tooth Explorer”3D visualization, was highly

appreciated and rated by students (Abdalla 2020).

9.5.9 Virtual and Augmented Reality

Digital applications are widely used in all areas of

dental education (Roy et al. 2017; Murbay et al.

2020). In addition to 3D, computer simulations

are showing promising results. Virtual Reality

(VR) is deﬁned as a computer-generated recrea-

tion of a world or scenario where the user

experiences the virtual world personally via sim-

ulation in real time (Joda et al. 2019).

Augmented Reality (AR) on the other hand is

deﬁned as a system that superimposes virtual

objects onto the physical world (Bölek et al.

2021). In a recent review published by Zitzmann

et al. exploring digital technology in dentistry

education, the authors conclude that VR and AR

will play a dominant role in dental education in

the future (Zitzmann et al. 2020). An example of

VR being used for dental morphology education

is presented by Liebermann et al. who developed

an interactive VR teaching environment that

utilizes a HTC Vive Pro headset (Liebermann

and Erdelt 2020). Within this environment dental

students were presented with a labeled 3D model

of the jaw with teeth and had the facility to select

individual teeth that could then be manipulated

via scaling, rotating, and moving. Additional

images, text, and audio were also available within

the environment to further reinforce the educa-

tional content. The authors evaluated their VR

system with 63 pre-clinical dentistry students via

a custom survey and found that the majority of

participants felt they understood dental morphol-

ogy better than when compared to traditional

textbook teaching (34.9% selecting “Much Bet-

ter,”and 57.1% Selecting “Better”). The authors

also found that students were willing to purchase

VR equipment privately to support their learning,

and that haptic and auditive elements were

evaluated more highly than just visual

(Liebermann and Erdelt 2020).

In the previous VR example, a specialized VR

headset was required to facilitate the delivery of

the educational content. However, as the potential

for VR and AR continue to grow, specialized

headsets are becoming cheaper, and VR/AR

environments can now be delivered via

smartphone applications. Juan et al. demonstrate

such an example whereby they developed an AR

smartphone app to support dental morphology

education (Juan et al. 2016). The AR app accesses

the smartphone camera which allows the student

to focus on a printed 2D image of a jaw, the app

then detects the image and displays a 3D model of

the jaw with teeth over the image. The student can

then choose a speciﬁc tooth to view in isolation

which can be further explored by scaling or

manipulating the camera location. There are addi-

tional learning aids within the app that overlays

all morphological details. The AR app was then

evaluated with undergraduate students, postgrad-

uate students, and academic staff, and

demonstrated that the app was effective in knowl-

edge transmission and for reinforcing acquired

knowledge (Juan et al. 2016). Furthermore, Haji

et al. (2021) conclude within their review of AR

for dental education that the beneﬁts of AR and

VR for dental education are evident and can fur-

ther provide access to quality educational

interactions with an overall lowered cost to train-

ing (Haji et al. 2021).

The combination of VR and AR is referred to

as Mixed Reality (MR), and despite its perceived

advantages in other dentistry applications, it has

yet to be used speciﬁcally for dental/tooth mor-

phology education (Monterubbianesi et al. 2022).

9 Visualizing Anatomy in Dental Morphology Education 197

9.5.10 The Web as a Learning

Technology

Harnessing the power of the web as a learning

technology is not a new or novel method; how-

ever, the increasing capabilities of the modern

web continue to support innovations in the deliv-

ery of the educational content. Early web

applications were limited and were mostly reﬁned

to the display of text, images, audio and video

ﬁles. However, advances in web technology

(namely HTML5) have allowed for the integra-

tion of interactive 3D, VR, and AR making it a

powerful CAL system. As regarded by Zitzmann

et al.,the possibilities of e-Learning can create

meaningful and enjoyable learning experiences

that are accessible anytime for dental students

(Zitzmann et al. 2020). However, researchers

and developers within this space often disagree

on domain deﬁnitions, as in other areas of tech-

nology/multimedia, as the potential and power of

the technology evolves, so too does its deﬁnition

(Moore et al. 2011), often narrowing or broaden-

ing the deﬁnition scope. For example, “E-

Learning”is considered by some researchers as

strictly web-based education, whereas others

include ofﬂine computer-assisted teaching aids

such as CDs/DVDs (Moore et al. 2011). For the

purpose of the below sub-sections, we refer to the

e-learning deﬁnition as the explicit use of the web

as a learning technology.

9.5.11 E-Learning

E-learning has several beneﬁts: access to a greater

variety of learning materials, ﬂexible learning

environment with control over the pace and

accessibility of learning, more adaptive than tra-

ditional teaching methods alone and offering

increased visualization. Additionally, it offers

educators with an easily updatable multimedia

platform for interactive teaching and improved

cognitive skills (Salajan et al. 2009; Wright and

Hendricson 2010; Maggio et al. 2012; Arevalo

et al. 2013; Patil et al. 2015; Chavarria-Bolaños

et al. 2020; Movchun et al. 2021).

The introduction of e-learning into the

dentistal curriculum has been assisted by techno-

logical advances (Mitov et al. 2010; Manogue

et al. 2011) and students choosing to study with

various online resources (Abu Eid et al. 2013).

Blended learning techniques are beneﬁcial for

students with different learning styles and have

demonstrated to produce better results (Pereira

et al. 2007) along with increased student satisfac-

tion (Reissmann et al. 2015) and enhanced com-

munication between students and teachers

(Wright and Hendricson 2010).

However, the use of e-Learning for tooth mor-

phology education does not need to strictly use

online systems. Goodacre et al. (2021) demon-

strate this through their combination of online

webinars with at-home wax-up of teeth. Over

the course of three weeks, students were required

to attend 11 webinars and complete ﬁve waxing

projects through images and videos available via

the online 3D tooth atlas. It was concluded that

this novel approach yielded very good results

from the students and that they effectively learned

the didactic aspects required for tooth morphol-

ogy; however, most of the students preferred

face-to-face lectures and lab sessions for learning

tooth morphology (Goodacre et al. 2021).

9.5.12 Learning Management System

(LMS)

Learning Management Systems (LMS) are virtual

learning environments that can simulate face-to-

face learning and also allow for the sharing, man-

agement, and automation of educational

resources (de Oliveira et al. 2016). Commonly

used LMSs include Blackboard (2022), Moodle

(2022), and Canvas (2022).

These systems have been utilized for the deliv-

ery of educational materials for a number of

years; however, as these systems are further

developed, their potential use for the delivery of

educational materials increases. This was further

exempliﬁed during the COVID-19 pandemic

where social distancing regulations drove the

need for virtual and remote learning. Some

researchers considered the imposed necessity as

an enormous opportunity for dental educators to

move toward and fully harness LMSs for dental

teaching/learning experiences (Chavarria-

Bolaños et al. 2020). This can be seen in the

work of Lone et al. (2021) who developed an

interactive tooth morphology module via the Can-

vas LMS. This module included video recordings

of lectures, lecture notes, links to live sessions,

and an interactive Fédération Dentaire

Internationale (FDI) grid, whereby students

could click on each of the notations and be

brought to either a 2D ﬂashcard or interactive

3D model of the selected tooth. The authors

conclude that the course was a feasible solution

during the pandemic as student results for that

year were in line with previous years (Lone

et al. 2021) (Fig. 9.4).

198 T. Vagg et al.

Fig. 9.4 Study tools provided for the Tooth morphology

module during the COVID pandemic. FDI grids accessing

3D models for permanent and deciduous dentition and 2D

ﬂash cards. The models included were from the

SketchFab™website and were prepared by the University

of Dundee (permanent dentition) and the University of

Michigan (deciduous dentition). The 2D ﬂash cards for

permanent dentition were obtained from Wheeler’s Dental

Anatomy

9.5.13 Social Media

Social Media Platforms (SMP) are websites and

applications originally developed to allow users

to connect and share information and content with

each other in a social context, via text, image, and

video posts (Kurian et al. 2022). However, the

ubiquity and usability of such platforms has led to

their widespread usage in education as tools to

facilitate teaching, learning, communication, and

collaboration (Mcandrew and Johnston 2012).

9 Visualizing Anatomy in Dental Morphology Education 199

The advent of SMPs during the 2000s and

their subsequent use in dental education includes

social networking sites such as Facebook and

Twitter (Arnett et al. 2013; Siqueira et al. 2021)

video sharing sites such as YouTube (Knösel

et al. 2011; Mukhopadhyay et al. 2014), informa-

tion repositories such as “wikis”(Salajan and

Mount 2012), audio/podcasting platforms such

as iTunes (Jham et al. 2008) and blogging

platforms such as WordPress (Mcandrew and

Johnston 2012). In more recent years, the devel-

opment of newer SMPs and their popularity with

the “social media”generation of students, along

with increased availability of internet and

smartphones, has led to a further increase in the

use of SMPs in dental education (Rajeh et al.

2021; Kurian et al. 2022). This includes platforms

such as photo- and video-sharing social network-

ing sites such as Instagram, Snapchat, and TikTok

(Nguyen et al. 2021; Rajeh et al. 2021), as well as

social messaging app like WhatsApp (Martins

et al. 2022).

The usage of SMPs within dental education is

varied and often echoes the original purpose of

these platforms for social connection and commu-

nication. For example, Siqueira et al. (2021) used

Facebook groups as a forum for peer-to-peer dis-

cussion regarding course content and also for

communication between faculty and students,

which they found to lead to increased engagement

(Siqueira et al. 2021). Similarly, in a scoping

review on the use of WhatsApp in dental educa-

tion, Martins et al. (2022) concluded that the app

is a useful tool for exposing students to informa-

tion and also for communication between

students and teachers (Martins et al. 2022). Fur-

thermore, the visual aspect of these platforms,

which allow the creation and sharing of content

and resources such as images, illustrations,

animations, and videos, can facilitate methods of

student learning as previously described in

sections on both conventional and innovative

visual methods for teaching tooth morphology.

YouTube is one of the more popular SMPs

utilized by dental students (Burns et al. 2020).

with a large number of dental education videos

publicly available (Knösel et al. 2011). As part of

a tooth morphology module redesign, Abdalla

(2020) integrated YouTube videos into the digital

delivery of the new module to host videos of

recorded lectures and detailed demonstrations of

tooth morphology waxing. The authors note

that recording this content and making it

available to students has an advantage over live

demonstrations since the videos can be replayed

and watched at the students’own pace

(Abdalla 2020). As well as standard videos,

YouTube is now capable of hosting interactive

3D animations and videos for use on smartphone

VR devices, and it has the potential to be

further integrated into dental morphology educa-

tion as an accessible VR system. However,

YouTube must be used or recommended to

medical and dental students with extreme caution

(Bosslet 2011) in order to guarantee the accuracy

of content and reliability of writers/content

creators (Knosel et al. 2011; Mukhopadhyay

et al. 2014).

A survey by Nguyen et al. (2021) at two dental

institutions in the USA found that students felt

Instagram was a useful tool for reinforcing tooth

morphology content as well as encouraging fur-

ther engagement with the material outside of class

(Nguyen et al. 2021). In this study, the education

content consisted of faculty-curated private

Instagram accounts which posted images, videos,

practice questions, and quizzes (Nguyen et al.

2021). However, there are also a large number

of public Instagram accounts focusing on dental

anatomy content, although (similar to YouTube)

a potential drawback to these public accounts is

lack of quality control and ensuring information

is correct (Douglas et al. 2019). Nevertheless, the

visual nature of the image- and video-sharing

features of Instagram and other SMPs seem to

be a good ﬁt to the teaching of visually-focused

ﬁelds such as dental anatomy and tooth

morphology (Douglas et al. 2019; Nguyen et al.

2021).

200 T. Vagg et al.

9.6 Digital Teaching: Some Points

to Ponder!

In the preceding sections, we examined the con-

ventional teaching of tooth morphology and the

use of visualization, as well as changes in digital

education that offer visualization to their learners.

There are, however, a few crucial factors to con-

sider before e-learning is implemented and digital

changes are made to a curriculum.

Before giving the ﬁnal takeaway message, the

authors of this chapter will outline and explore

several crucial and fundamental factors that need

to be considered for digital and online education.

9.6.1 The Digital Divide

ICT (Information and Communication Technol-

ogy) is changing how dental morphology is

visualized, as well as education in general, as

was previously stated. However, with these

advancements and potential implementations of

ICT in the Dental Curricula comes a “Digital

Divide”with a global disparity noticed, with

some institutes reporting high (Hamissi et al.

2013) and low (Postma et al. 2020) dental student

ICT. This gap or Digital Divide is frequently used

in the context of education to describe the

disparities in access to technology (hardware,

software, and the internet) imposed by a variety

of reasons, including social, economic, and geo-

graphic ones (Baig et al. 2019). In order to pre-

vent these students from becoming “digitally

excluded,”some universities attempt to provide

their students with help such as equipment loan,

minor ﬁnancing awards, and accessible labs/study

facilities. However, this provision also applies to

other factors, such as training access and

familiarity with digital literacy abilities for the

students (the “digi divide”) (Salajan et al. 2010).

As a result, teachers using cutting-edge digital

learning tools should make sure that their pupils

have access to all required resources (technology,

the internet, etc.), including digital training

(Khatoon et al. 2019).

9.6.2 Accreditation of Digital

Technology in Education

Different LMS and CAL systems have been used

to facilitate remote learning in various elements

of dental education that required visualization

during the COVID-19 pandemic (Azab and

Aboalshamat 2021; Lone et al. 2021). The asyn-

chronous tools, however, may be utilized less

frequently or even abandoned altogether (Crome

et al. 2021) as we transition toward a post-

COVID setting and synchronous face-to-face/tra-

ditional approaches are once again viable. But

there are still circumstances where these LMS

and CALs for remote learning might be useful,

such as during a new pandemic, a virus outbreak,

to support students who are susceptible/high risk/

unable to attend due to bereavement/sickness, and

during natural disasters like storms and ﬂooding

(Longhurst et al. 2020; Mishall et al. 2022; Nassar

and Rajeh 2022). Therefore, it is strongly advised

that researchers and educators continue to think

about these technologies in order to promote

independent or distant learning (perhaps using a

blended/hybrid method), especially in developing

countries (Baig et al. 2019).

9.6.3 Suggested Recommendations

for Educators to Implement

Novel Visualization Tools

Academics and educators are ﬁnding it challeng-

ing to offer time to update tooth morphology

(especially in senior clinical years), where it is

most needed, due to operational challenges

imposed by the growth in dental student numbers,

decrease in healthy extracted teeth which can be

used for teaching, and reduction in teaching

hours. Therefore, putting in place cutting-edge

visualization technologies to assist self-directed

revision might aid in resolving these problems.

9 Visualizing Anatomy in Dental Morphology Education 201

9.6.4 Educators to Offer Multiple

Modes of Representation

In order to enable learning for all the learners in a

single environment, the Center of Applied Special

Technology (CAST) in the United States devel-

oped the educational framework known as Uni-

versal Design for Learning (UDL) (About

Universal Design for Learning 2022). Educators

can utilize this educational framework’s three

guiding principles and 31 checkpoints while cre-

ating the learning outcomes of their modules/cur-

riculum and also executing the delivery of the

teaching for the module. UDL offers ﬂexible

instruction and seeks to engage and inspire all

students. Various methods of representation,

numerous means of action and expression, and

multiple ways of interaction and engagement are

among the three fundamental tenets of UDL.

Tooth morphology is a basic science subject

with numerous applications in the clinical years

for dental healthcare professionals. Therefore, it

is crucial to make sure that dental students retain,

recall, and comprehend the morphological

properties of all the teeth rather than acquiring

information by rote. We can develop resourceful

and informed learners by putting the UDL multi-

ple means of representation principles into prac-

tice and allowing dental students to visualize the

tooth morphology using a variety of teaching

tools like extracted teeth, plastic teeth, 3D teeth,

2D ﬂash cards, and lecture recordings. The

ﬂipped classroom model has been successfully

implemented in medical and dental education

and shows promising results in the dental anat-

omy classroom by empowering the students to

improve their cognitive and psychomotor abilities

while the teacher promotes and supports their

learning (Chutinan et al. 2018; Kellesarian 2018).

9.6.5 Educators to Be Involved

in the Development/Selection

of Teaching Tools

Additionally, participating actively in the creation

and assessment of digital tools is advised for

educators interested in integrating digital technol-

ogy into the teaching of dental morphology. This

can take the form of actively examining the open-

source information that is currently accessible and

advising students on accurate and appropriate

tools, or it can take the form of creating/

collaborating on the creation of a new visualiza-

tion resource.

9.7 Individualizing the Process

with One University’s Example:

Tooth Morphology Module

Teaching at University

College Cork

At University College Cork, students can enter

the Bachelor of Dental Surgery (BDS) program

through two different routes, either direct BDS

entry after ﬁnishing secondary education, or a

graduate entry program, where mature students

with a third-level biological sciences degree can

apply (BDSG). The BDS degree is a 5-year pro-

gram whereas the BDSG degree is a condensed

4-year program. The tooth morphology course is

taught in the second and fourth semesters of the

dental degrees (BDSG and BDS respectively) and

includes 15 hours of face-to-face interactive

lectures and 12 hours of practical laboratory

hours with ~2.5 contact hours per week. The

teaching is supported by the university’sLM

Canvas allowing student’s access to lecture/prac-

tical handout, learning recordings, online

resources, along with any communication about

the teaching. The practical sessions are held in the

laboratory where students are offered hands-on

approach to learning with a wide range of

teaching tools such as extracted human teeth,

high-quality plastic replica teeth set, online 3D

tooth morphology quiz, and a self-assessment

opportunity with extracted teeth. Students are

offered self-directed learning to study the dental

morphological features of permanent and decidu-

ous teeth with a lecturer facilitating the learning

process (Lone et al. 2019). The recommended

supporting textbook and atlas are Wheeler’s Den-

tal Anatomy Physiology and Occlusion (Nelson

2014)andIllustrated Dental Embryology, Histol-

ogy and Anatomy (Fehrenbach 2016).

202 T. Vagg et al.

Fig. 9.5 Timeline showing the tooth morphology teach-

ing in the 5-year curriculum at University College Cork.

(a) Year 1 and Year 2 of the dental curriculum, where

tooth morphology is taught with various teaching tools. (b)

Year 3, 4, and 5, where clinical dental modules need the

vertical integration of tooth morphology. (c) Inclusion of

digital technologies to allow tooth morphology revision

throughout the dental curriculum

Assessment for this module has two elements,

a formative spot examination and an end-of-mod-

ule examination. The formative spot exam is a

timed exam held in the lab with various stations

having extracted human permanent and decidu-

ous teeth which the students are asked to identify

and also chart its notation using either the Fédér-

ation Dentaire Internationale (FDI) or Palmer

notation system. Each station is timed for one

minute. The end of the module examination

consists of essay and multiple-choice questions

and is 1.5 hours in duration.

In the diagram below the authors have

demonstrated the various teaching aids used in

the ﬁrst 2 years of the dental curriculum to teach

tooth morphology. It has also been demonstrated

where the revision of tooth morphology can be

employed in the clinical years and which clinical

specialities it could be helpful for, thus showing

vertical integration of tooth morphology

(Fig. 9.5).

9.8 Best Practice Moving Forward

The best way to teach tooth morphology’s funda-

mental concepts is through conventional lectures

and practical sessions supported by a variety of

cutting-edge auxiliary teaching tools. Therefore,

moving ahead, a blended/hybrid teaching para-

digm should be used. These cutting-edge digital

tools must be introduced in order to supplement

dental students’learning. These tools work best

when used in conjunction with traditional teach-

ing techniques like lectures and labs. Numerous

readily available supplemental teaching methods

like online tools, and software programs may be

helpful in grasping certain components of the

curriculum, however they cannot replace direct

visual and tactile contact with extracted teeth

since in situ teeth are the ones that the students

treat primarily in clinical settings (Risnes et al.

2019). Offering multiple teaching pedagogies

will ensure active student engagement in the

learning process. Futhermore, these digital tools

can be easily used in the clinical years to allow for

vertical integration of tooth morphology within

the dental currciulum.

9 Visualizing Anatomy in Dental Morphology Education 203

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Flashcards: The Preferred Online

Game-Based Study Tool Self-Selected by

Students to Review Medical Histology

Image Content

Priti L. Mishall , William Burton, and Michael Risley

Abstract

Medical students use several supplementary

digital resources to support learning. Majority

of these supplementary resources enhance

learning by recall and repetition. A few

examples of these resources are concept

maps, ﬂashcards (FCs), and self-testing tools.

Traditionally, paper-based FCs are used in

higher education. The concept of paper-based

FCs is extended to the digital world in the form

of electronic/web-based FCs. The use of elec-

tronic/digital ﬂashcards has been reported to

review course material in the medical school

curriculum. Some of the medical school

coursework requires students to acquire visual

skills, for example, histology and pathology.

Students, who do not have prior knowledge of

the basic content on histology and pathology

struggle to identify microscopic tissues and

organs. Therefore, students look for other sup-

plementary resources to support visual

learning. Digital resources like Anki, Quizlet,

and Osmosis provide study tools that support

visual skills. A review of the literature

revealed only a few publications pertaining to

the use of digital testing tools for histology

education in medical school curriculum. In

the medical histology course at the Albert

Einstein College of Medicine (Einstein),

Bronx, NY, ﬁrst-year medical students used a

game-based platform (Quizlet) to review

image-based histology course content in the

form of four Quizlet study sets. Students

chose from six Quizlet study tools (Flashcards,

Learn, Speller, Test, Match, and Race/Gravity)

to review the image-based course material and

test their knowledge on accurate identiﬁcation

of histological images. The data on student

usage of study tools was tracked and analyzed

for 4 years (Graduating Classes of 2018 to

2021) to calculate: the total usage of the

game-based study tools (Flashcards, Learn,

Speller, Test, Match, and Race/Gravity) over

the period of 4 years, total percent usage over

4 years of each game-based study tools

(Flashcards, Learn, Speller, Test, Match, and

Race/Gravity) in each of the four Quizlet study

sets and to identify the preferred game-based

study tool. The data showed a consistent year-

on-year increase in usage of game-based study

tools by 50% (M=445 in 2018 compared to

M=849 in 2021). For the four Quizlet study

P. L. Mishall (✉)

Departments of Pathology & Ophthalmology and Visual

Sciences, Albert Einstein College of Medicine, Bronx,

NY, USA

e-mail: priti.mishall@einsteinmed.edu

W. Burton

Department of Family and Social Medicine, Albert

Einstein College of Medicine, Bronx, NY, USA

M. Risley

Department of Developmental & Molecular Biology,

Albert Einstein College of Medicine, Bronx, NY, USA

#The Author(s), under exclusive license to Springer Nature Switzerland AG 2023

E. Abdel Meguid et al. (eds.), Biomedical Visualisation, Advances in Experimental Medicine and

Biology 1406, https://doi.org/10.1007/978-3-031-26462-7_10

209

sets the percent usage of each study tool

Flashcards, Learn, Test, Match, Gravity, and

Speller was tracked and combined across the

four academic years. It was found that

Flashcards were used signiﬁcantly more fre-

quently than any other tool and this was

followed by Learn, Test, Match, Gravity, and

Speller ( p<0.0001 using chi-square). The

study concludes that ﬂashcards are the pre-

ferred study tool used by students to acquire

visual skills for identifying histological images

and could be incorporated when designing

online study tools.

210 P. L. Mishall et al.

Keywords

Medical histology · Game-based study tools ·

Flashcards · Visual skills

10.1 Introduction

Web-based self-testing resources are used by

students to improve recall and repetition.

Several online formative assessment tools are

available in the market like Anki, Quizlet, Study

Blue, and CRAM. These self-assessment

resources do not count toward students’grades,

hence ideal for formative assessments. These for-

mative assessments support multiple

opportunities to relearn and receive feedback

and prepare well for the summative assessments

that count toward the student grades. The self-

testing tools are based on the educational

principles of spaced repetition and spacing. Addi-

tionally, these self-assessment tools have an

intrinsic ability to self-test student knowledge

and provide students feedback on their perfor-

mance to promote self-directed and independent

learning. These self-assessment tools are used in

medical school coursework, especially for visu-

ally demanding subjects like histology or pathol-

ogy. These self-testing tools eventually become

an integral part of the hidden curriculum

(Vogelsang et al. 2018) Students who do not

have prior exposure to this new material use sup-

plementary resources to develop visual skills that

allow for repeated reviews to develop visual accu-

racy to identify microscopic images.

This chapter explores the use of an online

game-based study tool Quizlet by ﬁrst-year medi-

cal students. Students used Quizlet as a supple-

mentary resource to review histology image sets.

This game-based online platform allows students

to hone their ability to identify a wide range of

histology images. Students used the Quizlet plat-

form to repeatedly review image sets until

students were conﬁdent in correctly identifying

the images. This study does not have a control

and experimental group and the study does not

measure the impact of the use of resources on

knowledge gain and retention. The study focuses

on the usage of study tools for 4 years

(Graduating Classes of 2018 to 2021) to calcu-

late: the total usage of the game-based study tools

(Flashcards, Learn, Speller, Test, Match, and

Race/Gravity) for 4 years, total percent usage of

game-based study tools (Flashcards, Learn,

Speller, Test, Match, and Race/Gravity) in each

of the four Quizlet study sets over 4 years and

ﬁnally to identify the preferred game-based

study tool.

10.1.1 Learning Resources that

Support Knowledge Retrieval

Formative assessment is critical for learning. Fre-

quent review of the course content by repetition

and recapitulation supports successful knowledge

retrieval (Jurjus et al. 2014,2016; Routt et al.

2015; Taveira-Gomes et al. 2015; Sun et al.

2021; Tsai et al. 2021). Several resources are

used by students to enhance learning, for exam-

ple, notes (Back et al. 2016; Luo et al. 2016;

McAndrew et al. 2016; Trelease 2016; Javaid

et al. 2018), drawings (Bell and Evans 2014;

Gheysens et al. 2017; Greene 2018; Shapiro

et al. 2020), concept maps (Muirhead 2006;

Demirdover et al. 2008; Karpicke and Blunt

2011; Thomas et al. 2016; Chen and Allen

2017), and Flashcards (Abramson et al. 2002;

Reilly 2011; Golding et al. 2012; Al-Rawi et al.

2015; Deng et al. 2015). Historically, the fre-

quency of repetition and timing of repetition has

been a matter of research for centuries.

Ebbinghaus described in 1800 that it is easier to

remember information when it is studied multiple

times over a long-time span rather than studied

once or a few times in a short time span

(Ebbinghaus 1913; Al-Rawi et al. 2015). This

principle is applied to study tools like Flashcards.

For example, a company called SuperMemo, a

commercial Flashcard program implemented

spaced repetition by keeping track of the ideal

time to review material, optimized based on the

performance of the user (Al-Rawi et al. 2015). In

the last two decades, students are using numerous

online platforms to review/self-test study material

to supplement their learning. There are several

beneﬁts of online learning platforms:

10 Flashcards: The Preferred Online Game-Based Study Tool Self-Selected by... 211

(a) Learners have the ﬂexibility to use the tools

anywhere and everywhere.

(b) The platform usually automatically

randomizes the content before the test. This

ensures that students are learning the material

and not simply the alphabetical order of the

content.

to self-assess their learning. Students receive

immediate feedback on the progress of

learning. This allows students to monitor and

modify their learning.

(d) The online learning platform is a self-testing

device that supplements self-directed learning.

(e) The online learning platform provides an

opportunity for interactivity. Interactivity is

achieved by providing learners with

opportunities for repetition and self-

assessment through immediate feedback

(Reilly 2011)

(f) The online learning platform supports

retrieval-based practice (Deng et al. 2015)

and spaced repetition (Al-Rawi et al. 2015;

Deng et al. 2015)

(g) The online learning platform promotes repe-

tition and active learning (Chariker et al.

2011; Reilly 2011)

10.1.2 Supplementary Learning

Resources and Educational

Principles

Generation Z students use a number of online

supplementary resources like Socrative, Kahoot,

Quizizz, Quizlet, Quizalize, Mentimeter, Blooket,

Riddle, and Brainscape (Kharbach 2022). These

supplementary resources become integral

components of the hidden curriculum (Hafferty

1998). The learning content of majority of these

supplementary resources is student-generated and

not always vetted for content accuracy by the

expert faculty. The popularity of a self-directed

supplementary resource is based on the ability of

resources to allow students to self-test (Abramson

et al. 2002; Kornell and Son 2009; McAndrew

et al. 2016), spaced repetition (Kerfoot et al.

2007; Deng et al. 2015; Lu et al. 2021; Hart-

Matyas et al. 2019), and interactivity (Sweller

1994; Betrancourt 2005; Kirschner 2002; Back

et al. 2016; Holland et al. 2016). Spaced repeti-

tion allows learners to remember information

when it is done multiple times over a long time

span rather than studying once or few times in a

short time span (Fig. 10.1). Interactivity refers to

the process of a user utilizing input devices to

activate technology to elicit some type of visual

or audio response (Sims 1997). It is found that

interactivity plays a key role in knowledge acqui-

sition and development of cognitive skills

(Friedman 1996; Sims 1997; McLean 2001;

Patel et al. 2006).

10.1.3 Use of Digital Platforms

for Histology and Pathology

Teaching

Histology and Pathology courses report the use of

several online instructional programs to support

students’visual learning in the coursework.

Hoffman et al. (1992) developed “PathPics”to

help students process the large volume of visual

information and then use a quiz mode to assess

their mastery of material (Hoffman et al. 1992).

Some universities have made Virtual slides online

for students and faculty to view (http://www.path.

uiowa.edu/virtualslidebox/). Campos-Sanchez

et al. (2014) describe the use of a video as an

audiovisual learning notebook as a self-learning

tool in histology (Campos-Sanchez et al. 2014).

Michigan’s“eHistology—A SecondLook Series”

published a self-evaluation tool by students, who

want to test their level of preparedness before

taking quizzes and examinations (Hortsch

2013). Another study describes the student

preferences for an e-learning resource

SecondLook TM as a self-review histology tool.

The study compared three interfaces, PowerPoint

ﬁles, an online website, and a mobile application,

and concluded that “Convenience,”“larger

screen,”and “easy to use”PowerPoint ﬁles were

the most popular (Bringman-Rodenbarger and

Hortsch 2020). A couple of others report

institute-speciﬁc histology resources in the form

of images and accompanying quizzes (Brelje and

Sorenson 2014; Lisa and Oana 2022). Addition-

ally, student-generated and self-directed image-

based self-testing resources (for example, Anki

and Quizlet)are in demand to acquire visual

identiﬁcation skills in histology and pathology.

Anki decks present the image-based content in

only one testing mode that is as digital ﬂashcards

(Lu et al. 2021). On the other hand, the Quizlet

game-based platform presents learners with six

different testing modes (Flashcards, Learn, Test,

Match, Gravity, and Speller) to self-test the

image-based content.

212 P. L. Mishall et al.

Review

Key

Spaced repetition

Traditional study patterns

Time

Memory retention

Review

Initial Exposure

Review

Summative Exams

Fig. 10.1 Comparison of exposures to knowledge with

spaced repetition and traditional techniques. This diagram

illustrates the early focus of traditional learning techniques

on amassing practice, which creates an initially strong

memory that ultimately decays without further revision.

By comparison, spaced repetition is known to effectively

distribute meaningful practice across time to reinforce

learning and slow the decay of memory, promoting long-

term retention. “From Jape et al. (2022), originally

published under CC BY 4.0”

10.1.4 Rationale for a Study

in Histology Education

on Student Usage of Various

Study Tools Offered by

an Online Web-Based Platform

Quizlet

The study of medical histology requires the inte-

gration of structure with physiology and pathol-

ogy. Structure is mostly at the light and electron

microscope levels. Thus, medical students are

expected to gain mastery in identifying tissues

and organs by recognizing their salient features.

Most medical students have little prior exposure

or no exposure to microscopic image-based

learning. Students are trained to recognize normal

and abnormal structure on digitized histological

slides. These digitized images represent light or

electron microscopic views of the structures.

10 Flashcards: The Preferred Online Game-Based Study Tool Self-Selected by... 213

Many times, students fail to identify the organs

or tissues correctly due to struggles with develop-

ing appropriate visual cues. The goal of histology

courses is to ensure that students conﬁdently

identify organs and tissues by visualizing their

light and electron microscopic images. Students

in the histology course are trained to identify

hematoxylin and eosin (H&E) stained normal

histological tissues using visual cues. For exam-

ple, students learn to recognize the duodenum by

identifying the branched tubuloalveolar

Brunner’s glands in the submucosa or differenti-

ate a bronchus from a bronchiole by recognizing a

ring of hyaline cartilage in a bronchus compared

to the absence of cartilage in a bronchiole. First-

year medical students perceive learning and recall

of these varied microscopic images as very chal-

lenging (Garcia 2019). Therefore, students

demand resources that aid the development of

visual skills, especially a resource designed to

present microscopic images and test their ability

to correctly identify the image. Preferably, the

resource could repeatedly present diverse visual

images until the student gains conﬁdence in

correctly identifying the microscopic tissue in

the image. Hence, online supplementary study

resources become critical because these resources

allow students for repeated review of microscopic

images in a self-paced manner. Histology @Yale

is a well-designed web-based open lab resource

that has images with multiple-choice questions;

however, this resource is not accompanied by

gaming study tools (Takizawa 2011). To review

the existing published articles a PubMed and

MedEdPortal search using the term “image-

based quiz tools for histology education”yielded

zero and seven results, respectively. The

inclusion criteria were preclerkship histology

education, quizzing tools, e-learning, and self-

assessments. In the MedEdPortal, six articles

were excluded because of their focus on anatomy

and dermatology education. Only one histopa-

thology e-learning histology tutorial on lymph

node ﬁt the inclusion criteria. The authors of the

article used PowerPoint features to ask quizzes

but did not use an online quizzing platform (Soma

2016). None of these studies compared the use of

different study tools’effectiveness for self-

assessment in histology. Also, there is little data

as to which quizzing tool would be preferred by

students.

In the current study, students used the supple-

mentary resource on the Quizlet platform to

review histology image-based study sets that

provided more complete coverage of topics

included in the course, met diverse needs of

student’s example Visual, Aural and Read/

Write, and used current, relevant technology/

computer-assisted learning (CAL) that further

engaged interactive independent self-directed

learning. Additionally, the Quizlet program

allowed to track learner usage of the application.

However, none of the published literature reports

on the total usage of Quizlet study tools and the

preferred study tool self-selected by the learners.

10.2 Aims

First-year medical students at Albert Einstein

College of Medicine (Einstein) used six game-

based study tools namely Flashcards, Learn,

Test, Match, Gravity, and Speller offered by the

Quizlet platform to learn both light and electron

microscopy histology images. The goal of the

study was to determine the student usage of the

six game-based study tools to study image-based

histology study sets on the Quizlet platform. The

study aimed to investigate the usage of study

tools for 4 years (Graduating Classes of 2018 to

2021) to calculate: the total usage of the game-

based study tools (Flashcards, Learn, Speller,

Test, Match, and Race/Gravity), total percent

usage of each game-based study tool (Flashcards,

Learn, Speller, Test, Match, and Race/Gravity)

for each of the four Quizlet study sets and ﬁnally

to identify the preferred game-based study tool.

214 P. L. Mishall et al.

10.3 Methods

10.3.1 Participant Recruitment

First-year medical students for the Class 2018

(n=169), 2019 (n=172), 2020 (n=169), and

2021 (n=168) participated in the anonymous

retrospective study. The demographic data for

this larger cohort of students over a period of

4 years is similar. The study does not compare

between the gender differences or education or

prior knowledge of the participants. This was a

retrospective study approved by the institutional

review board of Einstein. All the participants

were deidentiﬁed.

10.3.2 Steps in Creation of Study Sets

Quizlet is an online platform that allows students

and teachers to create customized sets of learning

materials. Learners can review this study material

using eight different interactive study tools

namely Flashcards, Learn, Write, Spell, Test,

Match, and Gravity and Live. At Einstein

students did not use two study tools, namely

Write and Live. The study tool Write asked

students to type the name of the histological tissue

and the study tool Live allowed students to com-

pete live with students in another team in the

classroom setting. Note that the updated Quizlet

website in 2022 offers a fewer number of study

tools.

Considering student demand for a learning

resource in the ﬁrst-year medical histology course

at Einstein, Bronx, NY the authors assessed vari-

ous online quizzing platforms and decided to use

Quizlet because of its ease to upload histological

images and its relatively cheap annual subscrip-

tion rate and ability to maintain the privacy of

institutionally copyrighted content.

The authors used the online gaming platform

Quizlet to create four image-based histology prac-

tice sets. Most of the study sets on the Quizlet

website are free for all registered users. In the

current study, the authors purchased the annual

subscription-based Quizlet for teacher’s services

($34.99). This service enabled faculty to track

student usage of study tools and provided

students with an advertisement-free study experi-

ence. The four-histology image-based study sets

were created. Here are the steps to create the

image-based library. First, the correct name of

the microscopic tissue was typed, and the

corresponding icon of the microscopic image

was uploaded. These microscopic tissue images

were selected from a diverse library of light or

electron microscopic images. The left column

shows the text, and the right column shows the

corresponding histological image (Fig. 10.2).

Four Quizlet study sets were created. Students

used the study sets as a supplementary study

resource as the histology course progressed.

1. Quizlet 1: Cell structure and organelles, Epi-

thelium, Connective tissue, Bone, and

Cartilage

2. Quizlet 2: Muscle, Nervous system, Blood,

and Lymphatic tissue

3. Quizlet 3: Skin, Digestive, and Endocrine

4. Quizlet 4: Respiratory, Urinary, Male and

Female Reproductive systems

The number of histological images in each

study set and class is listed in Table 10.1.

The study sets were placed in a password-

protected Class folder on the website to prevent

tampering or inaccuracies. Only course faculty

and the Quizlet creator had access to the Class

folders and ability to alter the image-based study

sets. A speciﬁc Class link to the Quizlet website

was posted on the learning management system.

Students used the link to sign up for a free

account on Quizlet.com. After students created

an account, an auto-generated email request

from the student account was sent to the course

faculty administrator. Upon the administrator

accepting the request, the student received access

to the speciﬁc Class study sets. This mechanism

ensured that only registered students received

access to the institutionally copy-righted histolog-

ical images in the study sets. After gaining access

to the speciﬁc Class study sets, students had the

ﬂexibility to pick any or all the online study tools

to review image-based microscopic tissues and

hone their tissue identiﬁcation skills.

Study set

10 Flashcards: The Preferred Online Game-Based Study Tool Self-Selected by... 215

Fig. 10.2 Shows Quizlet 3 study set. The upper part of

the study set shows the title of the study set. The top right

corner displays the study tools offered to learners to self-

select. Then there are two columns. The left column shows

the correct name of the histological image, and the right

column shows the corresponding histological image. This

data of diverse images from light and electron microscopy

is saved in the library

Students used the Quizlet platform as a sup-

plementary learning resource on a voluntary

basis. The authors did not restrict students from

creating their own study sets or use existing his-

tology study sets that were freely available. Fac-

ulty did not track the names of individual students

to maintain anonymity. Students freely choose

any of the study tools offered by the Quizlet

website to practice identiﬁcation of the micro-

scopic images. Below is an overview of all

study tools:

The study tool Flashcards tests students’

knowledge by presenting a microscopic image

on the screen. Students use the space bar on the

screen to ﬂip the ﬂashcard and read the correct

answer. Students could also use the left and right

arrow keys to move between cards (Fig. 10.3a, b).

Table 10.1 The column on the left lists the number of Quizlet Study Sets and the column on the right shows the number

of images in each study set for the Class of 2018 and Classes 2019–2023

Number of images in each study set

Class 2018 Class 2019–Class 2021

Quizlet 1 74 53

Quizlet 2 85 85

Quizlet 3 56 56

Quizlet 4 44 44

The study tool Learn tests students’knowl-

edge of image identiﬁcation by tracking which

images the student correctly identiﬁed and what

images the student did not correctly identify; the

software then retests them of their mistakes.

The study tool Test provides customized prac-

tice tests on any study set by generating a random

test by using images from the study sets and

presenting the image identiﬁcation in multiple

format examples including matches, MCQs, and

true/false.

216 P. L. Mishall et al.

Fig. 10.3 (a) Study tool Flashcard. The screen shows an H&E-stained histological image. (b) Study tool Flashcard

(ﬂipped). The screen shows the correct answer for (a)

The study tool Match presents on the screen a

random display of histology images and the cor-

rect answers. The study tool Match challenges the

student to drag the correct answer and drop it to

the correct image by using the computer mouse.

The study tool Match additionally allows students

to compete with friends and attempt to beat the

current record. This activity encourages students

to achieve accuracy in image identiﬁcation in the

minimum amount of time (https://quizlet.com/en-

gb/help) (Fig. 10.4).

10 Flashcards: The Preferred Online Game-Based Study Tool Self-Selected by... 217

The study tool Gravity is like the classic space

invaders game where students try to prevent an

object, in this case an asteroid, from reaching the

earth by correctly entering the corresponding

name of the histological image in the text box.

The current study explores and discusses the

use of six study tools namely Flashcards, Learn,

Spell, Test, Match, and Gravity. At Albert

Einstein College of Medicine, students used

Quizlet as a supplementary learning resource

wherein students self-selected one or more of

these study tools to review the image content to

prepare for their formative assessments. The

Quizlet resource stored a library of histological

images aligned to the formative assessment quiz

topics in the course.

Data Analysis

Statistical analysis was performed using

Chi-square tests. Statistical analyses and interpre-

tation of data were conducted using the SPSS

statistical package, version 20 (IBM Corp,

Armonk, NY). This retrospective study was

approved by the institutional review board of

Einstein.

Data Protection

In this retrospective study, the data collection was

totally anonymous; no speciﬁc information which

may lead to the identiﬁcation of an individual was

released. Concerning the course evaluation ques-

tionnaire, participants may choose to skip any

question they did not want to answer. Appropriate

data security procedures and precautions were

adopted so that data obtained were kept secured

in a password-protected computer with access

only by the researchers.

At the end of the histology course, each year

quantitative and qualitative data was collected.

The quantitative data collection focused on stu-

dent use of each study tool. Also, students rated

the overall Quizlet experience using the Likert

scale (1–4). The qualitative data collection was

in form of student comments on the overall

Quizlet experience. The data was collected for

the following four Classes (Class 2018, 2019,

2020, and 2021). Statistical analyses were

performed, and p-values were calculated. The

statistical test used was a Chi-square homogene-

ity of variance test.

Results

1. The overall usage of the game-based study

tools Over the 4-year period.

The mean logins (M) on the Quizlet website

for the four Classes was as follows: Class of

2018 (M=445); Class 2019 (M=650); Class

of 2020 (M=955); and Class of 2021

(M=849), respectively (Fig. 10.5).

2. The percent usage of the study tools for each

Quizlet study set (n=4) over the 4-year period

For the four Quizlet study sets 1, 2, 3, and

4, the percent usage of each study tool

Flashcards, Learn, Test, Match, Gravity, and

Speller was tracked and combined across the

four academic years. It was found that

Flashcards were used signiﬁcantly more fre-

quently used than any other tool and this was

followed by Learn, Test, Match, Gravity, and

Speller ( p<0.0001 using chi-square)

(Fig. 10.6).

3. Identify the preferred study tool self-selected

by learners to review the histology image-

based content.

It was also found that the students’use of

Flashcards was signiﬁcantly greater compared

to their use of all other study tools combined

(Fig. 10.7). This was true for academic years

2018 ( p=0.004), 2019 ( p<0.0001), 2020

(p=0.007), and 2021 ( p<0.0001).

4. Student perception on the use of the Quizlet to

learn histology

In the course evaluations, students’response to

the Quizlet experience for the three graduation

classes were collected, except for the Class of

2020. Students responded to the statement

“The online Quizlet study tool aided my self-

assessment in studying histology.”The ratings

were as follows graduating class 2018 (3.86/

5), 2019 (3.3/4), and 2021 (3.5/4). The course

was evaluated out of 4 for all years except

2018. The qualitative data was analyzed, and

themes were identiﬁed; comments and

recommendations are in Table 10.2.

218 P. L. Mishall et al.

Fig. 10.4 Shows the study tool Match. The screen presents a random display of histology images and the correct

answers. The student is required to drag the correct answer to the corresponding histological image or vice versa

Fig. 10.5 The use of Quizlet tended to increase over the

4 years of the study, though use tended to decline within

each year. The x-axis shows the Quizlet study sets and the

y-axis shows total student usage (M =mean log-ins on the

Quizlet website)

10 Flashcards: The Preferred Online Game-Based Study Tool Self-Selected by... 219

Fig. 10.6 Flashcards were the most popular study tools.

The x-axis shows the different study tools while the y-axis

indicates the percent use of each study tool. Flashcard use

increased from Quizlet 1–3 as the course progressed and

remained high for Quizlet 4. It was revealed that within the

4 Quizlet study sets, Quizlet 3 reﬂected the maximum use

of the Flashcards study tool (70% of usage across all six

study tools)

Fig. 10.7 Flashcards became the preferred study tool over all other Study Tools combined. The x-axis shows the Quizlet

study sets. The y-axis shows percent usage of study tools

Recommendations by students

220 P. L. Mishall et al.

Table 10.2 Qualitative data in form of Student feedback to the question “The online Quizlet study tool aided my self-

assessment in studying histology”

Student comments on the statement “The online Quizlet study tool aided

my self-assessment in studying histology”

•Good for identifying organs and structures. •More general knowledge questions

•Provided collection of images with randomized order (instead of needing

to go through each lab or the textbook)

•Go over vocabulary

•Very nice supplementation to the course. •More images

•Really enjoyed it as a ﬁnal preparation before quizzes and tests to know

for sure that I know the material.

•More questions

•Helpful in practicing identiﬁcation. •Divide them into each topic and give

multiple chapters to study

•Excellent tool.

•Good resource

10.4 Discussion

10.4.1 Introduction

The current study demonstrated that the cohort of

preclinical ﬁrst-year medical students at a US

medical school who used the online game-based

learning platform Quizlet as a supplementary

resource to review histological image identiﬁca-

tion reported ﬂashcards as the most popular study

tool. The study demonstrated that in the duration

of 4 years the student use of ﬂashcards for all the

Quizlet study sets was higher than the combined

use of all other study tools: Learn, Test, Match,

Gravity, and Speller. There was an increase in the

use of Quizlet study sets each year, although,

there was a decrease in the use of Quizlet study

sets within the same year. Additionally, students

reported an overall higher satisfaction with the

use of Quizlet as a supplementary study tool to

review histological image identiﬁcations.

10.4.2 Use of Game-Based Study Tools

in Medical School

A number of studies reveal the use of supplemen-

tary learning resources in medical school Anki

(https://apps.ankiweb.net), Firecraker (2015),

Study Blue, 2011 (Chegg 2003), ALERT STU-

DENT platform (Taveira-Gomes et al. 2015).

Many studies have shown that interactive instruc-

tional techniques can increase student interest,

cognitive processing, and curricular integration

(Bills 1997; Leong et al. 2003; Gould et al.

2008; Khalil et al. 2010). The current study

found that the year-to-year use of Quizlet study

tools to review image-based content for the his-

tology course was increased by almost 50%

(M=445 in 2018 compared to M=849 in

2021). This increase can be attributed to

recommendations by senior-year students.

Although authors do not have data on prior expo-

sure of students to online game-based tools in

undergraduate college, it is highly likely that

students who might have previously used a

game-based study tool would tend to use similar

study tools in future for their learning purposes

(Erhel and Jamet 2013). Within each year, there

was a decline in usage of Quizlet, and that might

be related to familiarity with the course content.

As the course progressed students became famil-

iar with the learning resources that they may

prefer over Quizlet.

10.4.3 Flashcards Versus Other

Game-Based Study Tools

A few examples of digital FCs include Anki,

Cram, Open cards, Osmosis, and Quizlets (Hart-

Matyas et al. 2019). Flashcards were reported to

be commonly used in undergraduate colleges

(Kornell and Bjork 2008) and allied health pro-

fessional schools (Al-Rawi et al. 2015;

McAndrew et al. 2016; Wanda et al. 2016). A

couple of publications reported on the use of

ﬂashcards as a study tool in medical schools

(Allen et al. 2008; Taveira-Gomes et al. 2015).

None of these studies compared the use of

ﬂashcards to other study tools. The current study

compared student usage of ﬂashcards to other

study tools and reported that the study tool

Flashcards were used signiﬁcantly higher

(p<0.0001 using chi-square) and was followed

by other study tools Learn, Test, Match, Gravity,

and Speller. The present study is unique in com-

paring the use of ﬂashcards to other study tools

while the previously published studies reported

on electronic FCs as a singular study tool.

10 Flashcards: The Preferred Online Game-Based Study Tool Self-Selected by... 221

10.4.4 Flashcard Use and Popularity

in Higher Education

and Professional Training

Traditional paper-based ﬂashcards are popular in

undergraduate colleges (Golding et al. 2012). The

use of digital ﬂashcards was reported in a number

of training specialties; examples include a dental

hygiene school to study oral pathology (Al-Rawi

et al. 2015), graduate school to study molecular

and cell biology (Taveira-Gomes et al. 2015),

degree courses like pharmacy school to study

brand and generic drug names (Whitman et al.

2019) and medical school to study clinical

nephrology and psychiatry (Allen et al. 2008;

Schmidmaier et al. 2011; Deng et al. 2015) and

recently digital ﬂashcards were reported to be

used in postgraduate medical training like

OBGYN (Tsai et al. 2021) and Orthopedics

(Lambers and Talia 2021).

FCs work best with questions that require

lower-order thinking skills, such as memorization

of speciﬁc facts (Anderson and Krathwohl 2001;

Schmidmaier et al. 2011). O’Hanlon and Laynor

(2019) published a commentary on a new genera-

tion of online study resources and mentioned sites

like SketchyMedical and Picnomic that use visual

learning mnemonics (O’Hanlon and Laynor

2019). None of these publications reported on

the use of ﬂashcards for visual image-based

learning including histology in medical school

training. The current study reveals FCs as the

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