Role of Optical Spectroscopic Methods in Neuro-Oncological Sciences

Abstract

In the surgical treatment of malignant tumors, it is crucial to characterize the tumor as precisely
as possible. The determination of the exact tumor location as well as the analysis of its properties
is very important in order to obtain an accurate diagnosis as early as possible. In neurosurgical
applications, the optical, non-invasive and in situ techniques allow for the label-free analysis of
tissue, which is helpful in neuropathology. In the past decades, optical spectroscopic methods
have been investigated drastically in the management of cancer. In the optical spectroscopic
techniques, tissue interrogate with sources of light which are ranged from the ultraviolet to the
infrared wavelength in the spectrum. The information accumulation of light can be in a reflection
which is named reflectance spectroscopy; or interactions with tissue at different wavelengths
which are called fluorescence and Raman spectroscopy. This review paper introduces the optical
spectroscopic methods which are used to characterize brain tumors (neuro-oncology). Based on
biochemical information obtained from these spectroscopic methods, it is possible to identify
tumor from normal brain tissues, to indicate tumor margins, the borders towards normal brain
tissue and infiltrating gliomas, to distinguish radiation damage of tissues, to detect particular
central nervous system (CNS) structures to identify cell types using particular neurotransmitters,
to detect cells or drugs which are optically labeled within therapeutic intermediations and
to estimate the viability of tissue and the prediction of apoptosis beginning in vitro and in
vivo. The label-free, optical biochemical spectroscopic methods can provide clinically relevant
information and need to be further exploited to develop a safe and easy-to-use technology for
in situ diagnosis of malignant tumors.

Full text

51
Review Article
Journal of Lasers in Medical Sciences Volume 6 Number 2 Spring 2015
Role of Optical Spectroscopic Methods in
Neuro-Oncological Sciences
Maryam Bahreini
Laser and Plasma Research Institute, Shahid Beheshti University, G. C., Evin, Tehran, 1983963113, Iran
Abstract:
In the surgical treatment of malignant tumors, it is crucial to characterize the tumor as precisely
as possible. The determination of the exact tumor location as well as the analysis of its properties
is very important in order to obtain an accurate diagnosis as early as possible. In neurosurgical
applications, the optical, non-invasive and in situ techniques allow for the label-free analysis of
tissue, which is helpful in neuropathology. In the past decades, optical spectroscopic methods
have been investigated drastically in the management of cancer. In the optical spectroscopic
techniques, tissue interrogate with sources of light which are ranged from the ultraviolet to the
infrared wavelength in the spectrum. The information accumulation of light can be in a reflection
which is named reflectance spectroscopy; or interactions with tissue at different wavelengths
which are called fluorescence and Raman spectroscopy. This review paper introduces the optical
spectroscopic methods which are used to characterize brain tumors (neuro-oncology). Based on
biochemical information obtained from these spectroscopic methods, it is possible to identify
tumor from normal brain tissues, to indicate tumor margins, the borders towards normal brain
tissue and infiltrating gliomas, to distinguish radiation damage of tissues, to detect particular
central nervous system (CNS) structures to identify cell types using particular neurotransmitters,
to detect cells or drugs which are optically labeled within therapeutic intermediations and
to estimate the viability of tissue and the prediction of apoptosis beginning in vitro and in
vivo. The label-free, optical biochemical spectroscopic methods can provide clinically relevant
information and need to be further exploited to develop a safe and easy-to-use technology for
in situ diagnosis of malignant tumors.
Keywords: spectroscopy; neuro-oncology; optics
Introduction
Application of optical spectroscopic methods in
diagnosis of cancer has been an attractive area of
interest during the past decades. Optical spectroscopy
is dealing with a group of methods that provide the
structural or functional data from cells and tissues by
optical exploration. All types of optical spectroscopic
methods are dealing with light-tissue interplays. These
interplays can be utilized for data extraction of the
structure or chemistry of the investigated tissue. There
are various famous optical spectroscopy methods such
as diffuse reflectance, fluorescence, vibrational and
Raman spectroscopy. In fluorescence spectroscopy, a
tissue absorbs a wavelength of light and emits it at a
longer wavelength. This technique can be used to obtain
information about the endogenous fluorophores or an
injected fluorophore. In reflectance spectroscopy, a tissue
is illuminated by light which is scattered and/or directly
reflected back. Due to the alternations of morphology and
structure at the cellular and subcellular level, reflectance
spectroscopy can easily differentiates gray and white
Please cite this article as follows:
Bahreini M. Role of Optical Spectroscopic Methods in Neuro-Oncological Sciences. J Lasers Med Sci 2015;6(2):51-61
*Corresponding Author: Maryam Bahreini, PhD; Laser and Plasma Research Institute, Shahid Beheshti University, G. C.,
Evin, Tehran, 1983963113, Iran. Tel: +98-2129904018; Fax: +98-2122431775; E-mail: M_Bahreini@sbu.ac.ir

Optical Spectroscopy in Neuro-Oncology
52 Journal of Lasers in Medical Sciences Volume 6 Number 2 Spring 2015
matter 1,2. Another type of optical interrogation, Raman
spectroscopy, relies upon the fact that a small fraction
of light undergoes inelastic (Raman) scattering from
the tissue. The scattered light have slight shifts in the
wavelengths due to interactions between the incident light
and biochemical groups such as amide bands, methyl
groups, and ringed structures on subcellular structures
such as nucleic acids and proteins. More information
about the biochemistry of the underlying tissues may
be collected from Raman spectra 3,4.
Approximately, 2.3% of all deaths related to cancer is
due to the brain cancer 5. In order to provide optimal and
personalized therapy, it is crucial to precisely determine
localization and boundaries of tumors as well as their
exact properties and characteristics as early as possible
to adjust therapy accordingly. The most prevalent
neoplasms of the brain reported in adults are gliomas
and brain metastases 6. Gliomas demonstrate a diverse
group of brain tumors with signed intra- and inter-tumor
variation. They are the most common of primary brain
tumors, accounting for over 60% of all cases 1,7. In the
case of for instance high-grade malignant gliomas, it is
important to remove the overall tumors and to maintain
the surrounding functional brain 8. Malignant gliomas
are a severe pathology due to their invading features
and restricted intracranial region. MRI images before
operation can give the localization and size information
about the tumor. These images can be used for neuro-
navigation during surgery but they cannot compensate for
intra-operative tissue changes and alterations, eg, shifts 9.
Intra-operative MRI requires profound constructional
changes of the operating theatre, and special, expensive
equipment 10; it is time-consuming (15–30 min) and is
used to optimize the extent of resection 11; for resection
control and not during ongoing surgery. Information
about localization of certain tumor types can also be
obtained by fluorescence monitoring 12. Nevertheless,
there is no method available that allows the localization
of every type of brain tumor during surgery. The exact
normal and tumorous tissue delineation during surgery
is an unsolved problem. Additionally, it is not possible
to detect small micrometastases before the blood-brain
barrier breakdown with the technology which is clinically
available 13, 14. To optimize the resection strategy and
decide upon radical resection of aggressive and highly
malignant glioma, a biopsy sample of suspicious tissue
is removed and the morphology of tissue is appraised by
a trained pathologist 15. This method is time-consuming
(approximately 30 min) and it is only applicable after
removal of the tissue. On the other hand, neurosurgery
renders it possible to visually access the tumour and
therefore theoretically opens the possibility to perform
in situ diagnosis without tissue removal through the
application of non-invasive optical analysis of suspicious
tissue. New advanced optical methods allow the label-
free investigation of tissues and have the potential
for histopathological diagnosis 16 which can address
biochemical properties.
Therefore, the present paper focuses on the various
optical spectroscopic method for the pathology assessment
of brain cancer and novel methods for the in vivo
discrimination of margins of the tumor. In this paper,
we first discuss the advantages of optical techniques
in clinical oncology, the optical spectroscopy principle
and the common experimental setup. Furthermore,
we describe several optical spectroscopy methods in
diagnosis of cancer. Various famous optical spectroscopy
methods including diffuse reflectance spectroscopy,
fluorescence spectroscopy, vibrational spectroscopy
and Raman spectroscopy. Our focus in this review is
on optical spectroscopy in neuro-oncology 17-21. So, due
to relatively minor investigated in clinical applications,
the alternative optical spectroscopy methods including
light scattering spectroscopy 22, coherent backscattering
spectroscopy 23, and low coherence spectroscopy 24, are
not discussed. Some specific applications are discussed
in the other section.
Advantages of optical techniques in clinical
oncology
Nowadays, optical spectroscopic techniques can be
served as acceptable methods in comparison to other
imaging techniques including magnetic resonance
imaging (MRI), x-ray computed tomography (CT) and
ultrasonography in clinical oncology.
Optical techniques possess several advantages
compared to above mentioned modalities which cause
them to be acceptable in clinical management of cancer:
the irradiation in optical range is not ionizing and does
not cause a health problem even for a long time of
exposure; the optical experiments have sensitivity to
alternations of biochemistry and morphology that are
related to carcinogenesis; technical improvements in the
detectors and sources of light have made it possible to
reach to real time measurements in several conditions;
the fiber-optic probes development and miniaturization
of detectors has made it practical to combine optical
spectroscopy with systems of endoscopy to achieve to
interior organs 25.

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Journal of Lasers in Medical Sciences Volume 6 Number 2 Spring 2015
In general, the depth of penetration of light is small
in comparison with x-ray or ultrasound. However,
modulation of wavelengths of light can vary the sensing
depth of optical experiments from a several micrometers
to hundred centimeters 26. The tunability of sensing depth
can add extra flexibility and sensitivity for special clinical
applications.
Principles of optical spectroscopy for cancer
diagnosis
Several light tissue interplays such as absorption,
fluorescence and scattering are the basis of optical
spectroscopy for tissue characterization. When a specie
absorbs excitation light photons without radiating
any photons, absorption occurred. In the whole
optical spectrum, the major light absorber in tissues
is hemoglobin 27. Total concentration of hemoglobin
shows the vascularization degree, which is important in
testing angiogenesis within cancer progress. Depending
on the degree of oxygenation, the absorption spectrum
of hemoglobin can change. Therefore, hemoglobin
oxygenation is the other significant factor as well as
total hemoglobin concentration, which have effect on
the absorption of tissue. The hemoglobin oxygenation
changes could show the variations in the balance between
the need and the reserve of the oxygen in tissues while
the hemoglobin is the oxygen carrier in the blood. The
absorption of lipid and water is in near infrared region
of light. Several studies have represented that lipid and
water amounts are considerably distinguished between
normal and cancerous breast tissue 28.
Vibrational optical spectroscopy is relying on the
specific wavelength of light absorption via vibrational
levels of molecules. In case of biological samples and
tissues, which consist of a variety of different biochemical
compounds, vibrational spectra are the products of the
complex overlap of multiple bands of the chemical
bonds of all tissue constituents. Therefore, vibrational
spectra comprise the entire information about cell or
tissue biochemistry and are referred to as biochemical
fingerprint. Vibrational spectroscopic techniques therefore
have been gaining increasing attention for biomedical
applications, investigation of disease mechanisms, and
diagnosis over the past years 29, 30. Fourier-transform
infrared spectroscopy (FT-IR) is relying on the infrared
radiation absorption by the sample and its use in
biomedical science has been studied for many decades.
After interplaying with a molecule of the tissue,
when one photon is bend from the direction of incident,
elastic or inelastic scattering occurred. Elastic scattering
or Raleigh scattering is a kind of scattering which
happens without the alternation of frequency of the
photon. Nuclei, mitochondria and collagen fibers 31 are
the major tissue elastic scatterers. Due to the variations
in the size of nuclei, the ratio of nucleus to cytoplasm
and the collagen fibers density in cancer cells, elastic
scattering has been shown to be effective in detecting
epithelial pre cancers 32, 33. When the alternation in the
frequency happens, inelastic scattering took place. Based
on whether the reduction or addition of the frequency of
scattered photons, inelastic scattering is named Stokes
or anti Stokes Raman scattering 34. Raman scattering is
the basis of Raman spectroscopy. Collagen, water, the
nucleus and cytoplasm of the cell, fat and cholesterol like
lipid deposits are Raman scatterers in tissues 35.
When a molecule return from an excited singlet state
to the lower singlet state and emits light, fluorescence
occurred. The wavelength of the radiation is often
longer than the wavelength of excitation, because of the
dissipation energy in the action. When a fluorescence light
emitted from fluorophore molecules (molecules exhibiting
fluorescence), it subjects to absorption and scattering.
Flavin adenine dinucleotide (FAD) and nicotinamide
adenine dinucleotide (NADH) are the most extensively
studied fluorophore molecules which are metabolic
coenzymes in the action of reduction oxidation for energy
production for cellular functions. The fluorescence of
these two fluorophores has been investigated to explore
the metabolic rate change of tissues 36. The range of
wavelength in fluorescence spectroscopy is usually bigger
than that in Raman spectroscopy. In diffuse reflectance
spectroscopy, light intensity measurements are done at
the excitation wavelengths, which are sensitive to the
absorption and scattering features of the tissues.
Experimental Instrumentation
The main components of an optical spectroscopy
system are listed below:
1. A source of light (such as laser);
2. Wavelengths selection elements (such as a band-pass
filter) for the selection of both the illumination and
emission radiations;
3. A fiber for illumination light delivery and emission
light collection;
4. A detector for signal intensity measurements.
Xenon lamp is usually used to utilize a large range
of wavelengths in diffuse reflectance spectroscopy and
fluorescence spectroscopy. When high power excitation

Optical Spectroscopy in Neuro-Oncology
54 Journal of Lasers in Medical Sciences Volume 6 Number 2 Spring 2015
light is needed, lasers can also be used. Monochromator
can be used as dispersing element for detection that is
usually connected to a single-channel detector such as a
photomultiplier tube (PMT). Another dispersing element
is a spectrograph, which can be connected to a multi-
channel detector such as a charge coupled device (CCD).
Usually, the excitation source in Raman spectroscopy
is a high power laser, due to the weak Raman signals.
A filter with transmission of laser light wavelength and
suppression of other wavelength, is usually used. In the
detection section, a long pass or notch filter is applied
to remove the excitation wavelength.
Optical spectroscopy techniques in clinical
cancer diagnosis
As it was mentioned, various optical spectroscopic
methods can play important roles in many aspects of
clinical brain cancer. In this section, some applications
of these methods are reviewed separately.
Fluorescence and Diffuse Reflectance
Spectroscopy
Diffuse reflectance and fluorescence spectroscopy
have been investigated in the evaluation of tumor
margin or characterization of the tissue during
neurosurgical operation 37. The application of diffuse
reflectance spectroscopy in identification of tissue types
for improving the intracerebral guidance within deep
stimulation of brain has been investigated by Antonsson
et al 38. For various functional targets of the internal
globus pallidus (GPi), subthalamic nucleus (STN) and
zona incerta (Zi), diffuse reflectance spectroscopy
experiments were performed in 10 patients. There are
considerable discrimination between the white and gray
matter intensities which is at least 14% (P < 0.05) for MRI
and 20% (P < 0.0001) for spectral sorted information. The
use of diffuse reflectance and fluorescence spectroscopy
to discriminate pediatric neoplastic and epileptogenic
brain from normal brain in an in vitro experiment was
studied by Lin et al 39. Diffuse reflectance spectroscopy
was performed between wavelengths of 400 and 900 nm;
fluorescence spectroscopy was performed at 337, 360,
and 440 nm excitation wavelengths for every sample.
The spectroscopic results are in good correlation with
pathological results for classification of brain samples
abnormalities. Statistically significant differences (P <
0.01) were obtaied for both raw and normalized diffuse
reflectance and fluorescence spectroscopic data for three
different matter of neoplastic and normal white matter of
brain, neoplastic and normal gray matter of brain, and
epileptogenic and normal gray matter of brain.
Diffuse reflectance spectra were used for assessment of
the of optical nerve discrimination feasibilities by Stelzle
et al to find the foundation in a feedback control system
to improve nerve maintenance in maxillofacial and oral
laser surgery 2,40. In the range of 350–650 nm wavelength,
diffuse reflectance spectroscopy were performed on nerve
tissue, fat tissue, mucosa, skin, bone, muscle and cartilage
of ex vivo pig heads. Tissue identification was made using
principal components analysis (PCA) in addition to linear
discriminant analysis (LDA) to discriminate nerve tissue
from various kinds of soft and hard tissue in the facial
section by means of diffuse reflectance spectroscopy.
Nerve tissue was discriminated accurately from fat
tissue, mucosa, skin, bone, muscle and cartilage with a
78% specificity of over and a sensitivity of more than
86%. These results demonstrated the overall feasibility
of applying diffuse reflectance spectroscopy in remote
discrimination of nerve.
Fourier-Transform Infrared Spectroscopy (FT-IR)
Compositional information about the biochemistry of
nervous tissue, grey and white matter can be obtained
from FT-IR spectra. Bands in the region of 1000–1350
cm–1 are dominated by the vibrations of phosphate groups
and carbohydrates and mainly indicate the presence of
phospholipids, DNA/RNA, and carbohydrates in the
context of cells and tissue. Prominent bands of amide II
and amide I bond vibrations of proteins are recognized at
around 1550 and 1650 cm–1, respectively. Bands related
to C-H bond vibrations in lipids and proteins are found
in the high-energy region from 2800–3000 cm–1 41. Used
as an imaging technique, FT-IR can provide spatially
resolved image of distribution of tissue components.
For investigation of brain tumors, FT-IR spectroscopy
has been intensively used. Primary brain tumors as
well as brain metastases of peripheral tumors can be
localized and discerned from normal brain tissue with
high specificity 42-44. The type of primary tumor can
be identified in case of brain metastases 45. The main
components that allow differentiation of normal and tumor
tissues and tumor-grading are the tissue lipid content
and alternations related to nucleic acids 46. Furthermore,
collagen content and distribution of collagen subtypes are
changed in brain neoplasms which can be investigated
by FT-IR spectroscopy 47.
It is possible to gain some diagnostic information

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including the glioma grade, expression of hormones
or tumor vascularization 48-50. The pathologically
and clinically increased hormone production could
be extracted from the spectral information using
chemometrical analysis as well as tumor identification 49.
Chemometrical analysis can sort spectral data according
to similarities and differences 51. They can be used to
build colorful maps of the tumorous tissue 52.
Attenuated total reflection FT-IR (ATR FT-IR) is a
type of infrared spectroscopy technique which can offer
the advantage of measuring non-transparent samples
such as bulk tissue. This technique requires tight contact
between the sample of interest and the core of the ATR
crystal or a fiber optic probe in an endoscopic setup 53.
Infrared radiation propagates in the crystal, generating
an evanescent wave that penetrates a few micrometers
of the sample. Spectral changes in the backscattered
light are used to obtain information about the sample’s
biochemical properties. This is especially interesting for
direct analysis of biopsy tissue 54. Using fiber optics,
it holds great potential for future clinical application
and in situ diagnosis of malignant glioma. The use of
optical ATR FT-IR spectroscopy for the analysis of native
human brain tumor biopsies indicates the possibility of
this method to find differences of extracellular matrix
components among different tumor types.
Raman Spectroscopy
In comparison to FT-IR spectra, in the Raman spectra
vibrational bands are better separated. So, the spectra
contain a higher degree of information. It is worth to
mention that the technique can be applied on non-dried
tissue because the spectral contribution of water does
not interfere with relevant bands of biological tissue as
it does in FT-IR spectroscopy 55. So, this property makes
Raman spectroscopy suitable for in situ diagnosis.
Raman spectroscopy of tissue permitted the
discrimination of healthy and tumor and necrotic tissues
in rat brain tissue samples 56 and was used to study
brain functions in living mice and rats 57. Brain injury
caused by traumatic insults related to caspase-3-activated
apoptosis can also be detected by Raman spectroscopy 58.
Raman mapping can identify brain tumors in the living
animal 59. Ex vivo studies on human brain tumor samples
have proven the capability of the method to distinguish
normal and tumorous tissues of adults and children 60-62.
Raman micro-spectroscopy of primary brain tumors can
provide diagnostic information on the malignancy grade
and cell density 63.
Raman spectroscopy was carried out for in vivo
mapping the surface of intercerebral tumors of rat brains
and comparison with hematoxylin-and-eosin (H&E)
stained coronal parts of the same area by Kirsch et
al 59. A multivariate chemometric method of k-means
cluster analysis of the spectra was applied for colorful
map construction of the rat brain surface. Surprisingly,
the mapping by Raman spectroscopy could find a tumor
below the surface that was not evident and cognizable
in photomicrographs visually.
In another recent study, Raman spectroscopy was
applied for the assessment of C6 glioblastomas implanted
in rat brains by Beljebbar et al 64. At first, Raman
spectroscopy was applied for classification of tissue cut
from the implanted brain tumors. The classification was
based on a set of reference spectra acquired from purified
DNA and lipids. The comparison was done between the
Raman spectroscopy results and those from conventional
histopathology as the classification standard. Principal
component analysis (PCA) was applied as a chemometric
method on the spectra acquired with 100% accuracy
for classification. It was assessed that reducing the
acquisition times from 100 s to 10 s had small impact on
the signal averaging while obtaining a robust procedure.
For demonstration of possibility of clinical utilization,
by means of a handheld Raman microprobe, implanted
tumors were investigated in vivo over 20 days. A distinct
discrimination of spectra of normal tissue before tumor
implantation from spectra acquired on days 4 and 20
after implantation was observed using hierarchical cluster
analysis. The immunohistochemistry staining and the
spectra were strongly correlated. Spectral signs were
correlated with invasive and proliferative characteristics
of the tumors. It is significant to find such spectral
signs that can play an important role in diagnosis the
biochemical change between cancerous and normal brain
tissue 65.
The possibility of spectral signs determination
relating to the lipids concentrations in the tissues was
examined using Raman spectroscopy of lipid extracted
from malignant tumors and normal tissues of brain. The
concentration variations in the of phosphatidylcholine and
cholesterol were significant between normal and cancerous
cells 66. Raman spectroscopic results of the lipid extracts
were in good agreement by mass spectrometric data.
Discrimination between neural crest-derived pediatric
tumors were investigated using Raman spectroscopy 67, 68.
Raman spectroscopy of freshly resected tissue or biopsies
obtained from 39 patients were acquired and analyzed
by PCA statistical method. For discrimination between

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56 Journal of Lasers in Medical Sciences Volume 6 Number 2 Spring 2015
nerve sheath tumors, neuroblastomas, ganglioneuromas,
pheochromocytomas and normal adrenal glands, good
sensitivity and specificity was attained 67.
For ex vivo Raman spectroscopy of fresh human tumor
samples, grading of astrocytoma was proved using fiber
optic probes 69. There are many researches that focus on
the application of Raman spectroscopy to perform optical
biopsies for tumor recognition 63. Technical advances in
the development and miniaturizing of Raman fiber probes
may allow short acquisition times of approximately 10
s in concert with high-quality spectra acquisition 64,70.
Raman endoscopy has already been performed in a clinical
context (> 300 patients) for gastric cancer diagnosis and
provided diagnostic information 71.
In order to increase the sensitivity and also reduce
acquisition time, different techniques are used to enhance
Raman signal intensity. A famous enhancing technique
is surface-enhanced Raman scattering (SERS). This
method exploits the electrochemical interaction of
molecules adsorbed by nanostructures. Raman signal
enhancements as much as approximately 1010 can be
achieved by putting the sample onto a suitable surface.
This technique is applicable for the analysis of chemical
substances or single cells, but not for large tissue samples.
Additionally, nanoparticles and compounds that exhibit
strong SERS signals were employed as alternatives to
fluorescent or colorimetric markers. In this context,
spectroscopy is not used to reproduce tissue properties but
to detect and reproduce the distribution of experimentally
introduced compounds in a sample. This can be used for
the detection and research of cancer and other diseases to
visualize the distribution of known markers detected by
classical immunohistochemistry 72. Another enhancement
technique is resonance Raman spectroscopy. If the energy
of the beam of the exciting laser approaches the optical
band gap of a tissue constituent, selected by appropriate
tuning of the excitation wavelength, the amplification of
the Raman signal takes place. The Raman signal intensity
is increased around 1000-fold and the resulting Raman
spectrum is dominated by the bands of the resonance-
enhanced molecule. So, detection of specific compounds
at very low molecular concentrations, such as NADH,
flavins, collagens, carotenoid, elastin and the heme
proteins can be done 73.
Some Specific Applications
Detection of Pediatric brain tumor
The utility of diffuse reflectance spectroscopy to
discriminate intraoperatively between pediatric tumors
and normal parenchyma of brain at the edge of resection
cavities was evaluated using an in vivo human experiment.
Diffuse reflectance spectra were obtained from normal
and tumorous brain areas of 12 pediatric patients during
their tumor resection procedures, using a spectroscopic
system with a hand-held optical probe. Statistical methods
were used and the results showed that diffuse reflectance
spectral intensities between 600 and 800 nm are effective
in terms of differentiating normal cortex from pediatric
brain tumors. Furthermore, probe movements induce
large variations in spectral intensities between 400 and
600 nm 74.
Infiltrating Tumor Margin (ITM) in Brain
Intraoperative identification of brain tumor margins
using optical spectroscopy was studied in a pilot clinical
trial of brain tumor of 26 patients. Diffuse reflectance and
autofluorescence spectroscopy was used for identification
of brain tumors and infiltrating tumor margins (ITM).
Using autofluorescence and diffuse reflectance at 460 and
625 nm wavelengths, a two-steps empirical discrimination
algorithm was constructed with 100% of sensitivity and
76% of specificity in discriminating of ITM and normal
tissues of brain. The contamination of blood was the main
difficulties that decrease the brain tumor demarcation
accuracy using optical spectroscopy. Generally, this study
indicates the feasibility of optical spectroscopy to conduct
the resection of brain tumor intraoperatively with great
sensitivity 2.
Many attempts at the use of optical systems in surgical
resection of gliomas have relied upon the introduction
of an exogenous fluorophore such as 5-aminolevulonic
acid (5-ALA). Where the blood-brain barrier (BBB)
breakdown has happened, 5-ALA is got by gliomas but
not in normal brain 75, 76. As a preliminary step of using
optical spectroscopy, one type of portable, handheld
system has been constructed and employed recently
which can be used to obtain spectroscopic data quickly,
and nearly real-time, in the operating room 37. In one
clinical trial for gliomas, spectral data of 24 patients
with glioma and 11 patients with mesial temporal lobe
epilepsy were acquired, in whom the pathologically-
normal temporal cortex was used as control brain tissue.
Results of the combination of diffuse reflectance and
fluorescence spectroscopy obtained 80% sensitivity
and 89% specificity in distinguishing solid tumor from
normal tissues. Furthermore, infiltrating tumor margins
were discriminated from normal tissues with 94%

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Journal of Lasers in Medical Sciences Volume 6 Number 2 Spring 2015
sensitivity and 93% specificity 77. These results suggest
that it may be possible to develop an “optical biopsy”
tool. A new optical spectroscopy probe compatible with
a biopsy needle has been designed and has completed
preclinical testing. A clinical trial is underway to examine
the efficacy of the smaller probe as a surgical adjunct
in stereotactic brain biopsy. It may be possible for these
tools to prepare the surgeon with near real time feedback
on the adjacency of tumor remnants. These techniques
may develop the percentage of resected tumor, in the
way that has been suggested in preliminary studies
with 5-ALA described above. Additional advantages of
these systems are that they are portable, fast, and easily
put into the operative field. These inexpensive systems
can reduce the need for more costly surgical adjuncts
including intra-operative MR (iMR) and multiple frozen
section tissue analyses 37.
Optical Spectroscopy of Radiation Necrosis
A common clinical problem in patients with malignant
gliomas who have been under intensive adjuvant therapy
is the development of new masses 78. The lesions may
demonstrate the recurrent of tumor, radiation necrosis,
or a combination of tumor and radiation injury.
Existing imaging methods including positron emission
tomography (PET) and magnetic resonance spectroscopy
(MRS) are often unable to discriminate recurrent tumor
from radiation necrosis 79, 80. Therefore, tissue biopsy
stays the gold standard for clinical decision-making.
Unfortunately, even needle biopsies of new areas of
contrast enhancement are confounding due to difficulties
related to sampling error 81. Recently, a novel spectral
feature was encountered in patients with radiation injury
and radiation necrosis 82. A shift of the predominant
spectral peak, the fluorescence peak at 460 nm, 40 nm
to the right has been identified as a hallmark of radiation
injury to the central nervous system. A special peak has
only been observed in patients with prior radiotherapy.
Absence of this peak has a 94% positive predictive value
in ruling out radiation injury of the cerebrum. These
spectral characteristics may be exploited to improve the
yield of stereotactic biopsies.
Optical Spectroscopy for Neuro-Navigation
Optical spectroscopy can also be applied for
identification of specific brain structures, such as nuclei,
which can be important in neuro-navigation. Diffuse
reflectance and fluorescence spectra were obtained from
cat brain in vivo to identify the optical and fluorescence
characteristics of various anatomical components
encountered on a trajectory from cortex through midbrain.
A representative two-dimensional plot of a set of diffuse
reflectance and fluorescence spectra was recorded from a
single interrogation path. By comparison, it can be found
that a depth-dependence in intensities and line-shape
variations in fluorescence and diffuse reflectance spectra
exist, which correlate with the anatomical structural
encountered along the interrogation pathway 37.
In addition, spectroscopic techniques have been used
in vivo to detect the excitatory amino acids glutamate and
aspartate 83, 84. Several neurotransmitters and precursors
are endogenous fluorophores. Fluorescence can detect
the characteristics of dopamine and other metabolites
in brain and tumor, which may have utility in guiding
surgical navigation, through metabolite detection as
well as by assessing the effects of therapy, especially if
certain metabolites, neurotransmitters, and endogenous
fluorophores are produced in response to anti-tumoral
agents 37.
Cell and Tissue Viability
Nicotinamide adenine dinucleotide and nicotinamide
adenine dinucleotide phosphate [NAD(P)H] has long
been considered as a dominant fluorophore of tissue,
with an emission wavelength peak at 450-470 nm. Optical
measurements of NAD(P)H have been used for decades
to interrogate cellular physiology due to its primary role
in oxidative phosphorylation and aerobic respiration 85.
In addition, NAD(P)H is involved in nucleotide donation
for DNA repair and is consumed during active cell death
during poly (ADP-ribose) polymerase (PARP) cleavage 86.
Optical Spectroscopic methods may be useful to rapidly
assess the utility of promising agents or novel techniques
in the treatment of neurological disorders. For example,
an in vivo fluorescence spectroscopy probe system that
can be implanted to monitor relative NAD(P)H levels
during therapeutic interventions have been developed 37.
Continuous or intermittent monitoring could allow the
identification, in near-real time (within minutes to hours,
instead of days to weeks) of response to a therapeutic
intervention. In animal models of tumors, for example,
if there were no change in the NAD(P)H levels, it would
be likely that this therapy was not producing cellular
killing and would likely be ineffective.
Using the example of malignant brain tumors, a patient
is usually treated with chemotherapy for 2-3 months prior
to obtaining a MRI to assess the utility of treatment. If

Optical Spectroscopy in Neuro-Oncology
58 Journal of Lasers in Medical Sciences Volume 6 Number 2 Spring 2015
the tumor has grown, the agent is abandoned and another
chemotherapeutic regimen begun. Using a minimally-
invasive optical spectroscopy device, information
regarding the efficacy of chemotherapy as measured by
NAD(P)H autofluorescence could be achieved within
a few hours or days rather than awaiting the next
MRI, several months later. Thus, optical spectroscopy
could become an effective means of identifying useful
chemotherapies tailored to individual responses 37.
Conclusions
Optical spectroscopic methods have been reviewed as
a useful in vitro/in vivo method in the neuro-oncology
science. Some specific applications including detection
of glioma tumor margins, identification of cerebral
radio-necrosis, and assessment of tissue viability, are
also discussed. As a future prospective application,
Optical spectroscopy has the ability to be a useful method
for monitoring the therapeutic response in a variety
of neurological conditions that rely upon cell killing
(neoplasia) or tissue survival (stroke, neurodegenerative
diseases). Furthermore, optical spectroscopy has the
potential to track a new generation of fluorophores for
surgical navigation, monitoring delivery of therapeutic
agents, and pharmacokinetic studies. These developments
may permit the rapid development of commercial
optical spectroscopic tools useful for neuro-oncology
in a variety of clinical applications. However, there is a
need for continual improvement of optical spectroscopic
instrumentation in order to accomplish at the necessary
level for clinical applications.
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