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Abstract
Infrared absorption spectra of formalin-fixed, paraffin-embedded human cervical tissue are reported for normal, dysplastic and neoplastic samples. The spectral differences found in this study between these states of the tissues are far less than those observed for single cells by us and others. Nevertheless, we find a direct correspondence between spectral data from tissue sections, obtained from biopsies, and individual exfoliated cells, typically obtained during a pap procedure. We also find that spectra due to dysplastic samples fall about halfway between the spectral features of normal and cancerous samples.
... Spatially resolved Infrared Microspectroscopy (IR-MSP) of tissue thin sections, in combination with digital imaging techniques, shows a great promise for in-vivo and ex-vivo medical diagnosis [1][2][3] . Since infrared spectroscopic methods are sensitive to the chemical composition of histological samples, changes related to diseases may be observed as small variations in chemical composition of cells and tissues. ...
... Since infrared spectroscopic methods are sensitive to the chemical composition of histological samples, changes related to diseases may be observed as small variations in chemical composition of cells and tissues. These changes may be translated into false color images by algorithmic methods that can be interpreted by non-spectroscopists, and are directly comparable to outcomes of standard histological staining protocols 3,4 . ...
... This method produces a large hyperspectral data cube, which may be represented as a series of chemical images at distinct wavelengths or as a matrix of spectra as a function of spatial position. However, data in this format are not useful for correlation against standard histopathology, or rendering medical diagnoses 3,4 . Thus, a number of methods are introduced that demonstrate how the data cube can be converted into two-dimensional images that present the spectral information in a format comparable to visual images obtained from corresponding stained tissue sections. ...
FT-IR microspectroscopy in combination with digital image reassembling methodologies have been used to characterize distinct tissue structures within thin sections from the human prostate. The spatially resolved microspectroscopic data were collected with a resolution near the diffraction limit (about 8 μm) by using a HgCdTe focal plane array detector-based infrared imaging instrument. While IR imaging based on distinct spectral parameters such as intensities, frequency values, or half-widths (the univariate imaging technique of chemical mapping) often gives unsatisfactory results, multivariate data analysis techniques (e.g. hierarchical clustering or principal component analysis) confirmed standard histopathological techniques and turned out to be helpful to discriminate reliably between different tissues structures.
... Spatially resolved Infrared Microspectroscopy (IR-MSP) of tissue thin sections, in combination with digital imaging techniques, shows a great promise for in-vivo and ex-vivo medical diagnosis [1][2][3] . Since infrared spectroscopic methods are sensitive to the chemical composition of histological samples, changes related to diseases may be observed as small variations in chemical composition of cells and tissues. ...
... Since infrared spectroscopic methods are sensitive to the chemical composition of histological samples, changes related to diseases may be observed as small variations in chemical composition of cells and tissues. These changes may be translated into false color images by algorithmic methods that can be interpreted by non-spectroscopists, and are directly comparable to outcomes of standard histological staining protocols 3,4 . ...
... This method produces a large hyperspectral data cube, which may be represented as a series of chemical images at distinct wavelengths or as a matrix of spectra as a function of spatial position. However, data in this format are not useful for correlation against standard histopathology, or rendering medical diagnoses 3,4 . Thus, a number of methods are introduced that demonstrate how the data cube can be converted into two-dimensional images that present the spectral information in a format comparable to visual images obtained from corresponding stained tissue sections. ...
FT-IR microspectroscopy in combination with digital image reassembling methodologies have been used to characterize distinct tissue structures within thin sections from the human prostate. The spatially resolved microspectroscopic data were collected with a resolution near the diffraction limit (about 8 micrometers ) by using a HgCdTe focal plane array detector-based infrared imaging instrument. While IR imaging based on distinct spectral parameters such as intensities, frequency values, or half-widths (the univariate imaging technique of chemical mapping) often gives unsatisfactory results, multivariate data analysis techniques (e.g. hierarchical clustering or principal component analysis) confirmed standard histopathological techniques and turned out to be helpful to discriminate reliably between different tissues structures.
... The unique frequencies of major molecular groups in biological tissues and cells have been characterized which the changes in the spectra have been shown to give malignant cells their characteristic histologic appearance [4]. Therefore, recently inspire by those outstanding results, there are many researchers who explore and apply FTIR spectroscopy as a diagnostic tool for the distinction between normal and malignant tissues and cells of several human cancers—including esophagus [5], colon [6], [7], skin [8], gastric [9], lung [10], gliomas [11], [12], and cervical cancer [13], [4], [14], [15], [16], [17], [18], [19]. As known that based on Bethesda System, cervical cells are classified into normal, LSIL, and HSIL. ...
... Although the neural network has been applied in a number of researches as intelligent tool for cervical cancer diagnosis, nevertheless they have not yet applied the features extracted from IR spectrum of cervical cells for classification process. Several researchers have investigated prominent features to distinguish the cervical cell conditions whether normal or abnormal cells [13], [4], [14], [15], [16], [17], [18], [19]. They have investigated peak of chemical bond bands of cervical cell spectra in certain regions. ...
Fourier transform infrared (FT-IR) spectroscopy is an effective tool for investigation of chemical changes at the molecular level. In cervical cells spectra, FTIR can reliably distinguish multiple types of cells. Our aim is to classify cervical cell biopsies into normal, low grade squamous intraepithelial lesion (LSIL), and high grade squamous intraepithelial lesion (HSIL) through the use of FT-IR spectroscopy using neural network. For classification, this study proposes new features of cervical cell spectrum that are suitable and can be used as inputs for neural network. The new cervical cell features are extracted from ThinPrep® spectrum and their applicability are tested by using Hybrid multilayer perceptrons (HMLP) network. All proposed features provide high accuracy with percentage over than 90 % in classifying the cervical cell into normal, LSIL and HSIL. These results show that all the proposed features are suitable to be used as input to neural network system for cervical cancer diagnostic system.
... 78 Other cell types and cervical debris were found to confound spectral diagnosis. 16,17,73,78 Because the spectral profiles for different cells can be similar, multivariate approaches were applied to resolve ''hidden'' structure within the data. These techniques enable spectra to be classified according to spectral variance and showed the potential for nonsubjective diagnosis, removed from the bias of human interpretation. ...
... 1,24,47,65,66 For this reason, it is better to confirm diagnosis using the gold standard biopsy and compare these results with FTIR analysis. Chiriboga et al 17 were the first to apply FTIR spectroscopy to cervical biopsies. They demonstrated that different spectral profiles could be obtained for the different cell types throughout the tissue section. ...
Fourier transform infrared imaging spectroscopy is a powerful technique that provides molecular and spatial information at the single-cell level. We report on the progress of this technology in the field of cancer research, focusing on human cervical cancer because of the inherent difficulty in grading this type of cancer and as a model for venereal cancers in dogs. Using a suite of multivariate imaging processing techniques, we demonstrate the potential of this technique to identify histologic features in the normal epithelium and cervical intraepithelial neoplasia stages I and III. We highlight the advantages and detail the barriers that need to be overcome before implementation of this technology in the clinical environment.
... The ® rst approach taken was that of correlating carefully collected tissue spectra with a detailed pathological analysis of the tissue architecture. 4,9 The second approach is to analyze the data strictly m athematically by per for m ing so m e d iscr im inant analysis and obtaining a picture of spectral relationships that retroactively can be correlated with pathology. ...
... Figure 7 shows spectral patterns in the DNA/ RNA spectral region, obtained from tissue biopsies, for normal cervical basal cells, as well as cervical basal cells diagnosed with dysplasia and neoplasia. 9 Befor e such sp ectr al changes can be interpreted as being due to cancerous disease, other confounding factors need to be eliminated. F or exam p le, architectu ral changes in the tissue m ay cause one to compare noncorresponding tissue types; furtherm ore, the body' s immune response may increase the num ber of lymphocytes or leukocytes in the diseased tissue. ...
An account is given on the state of the art in the interpretation of infrared microscopy of cells and tissues. Focus is on the subtle changes in the DNA/RNA spectral regions between normal, precancerous, and cancerous samples. This discussion aims at establishing the limits of infrared spectroscopy as a tool to diagnose cellular abnormalities that may be related to cancer.
... Then, the tissues were cut at 6 lm and stained with H&E and glial fibrillary acidic protein (GFAP). Supplemental tissue sections were mounted on a Au/ Ag layer slide that allowed spectra to be recorded in the transflection mode (Chiriboga et al. 1998). ...
Tissue analysis by Fourier transform infrared (FTIR) imaging can determine the biodistribution of molecules, without pre-analytical modification. We aimed to study the infrared spectroscopic changes of α-helical proteins at post-traumatic epileptic (PTE) foci by FTIR. FITR mapping was applied to detect α-helical proteins in rat brain tissue samples with post-traumatic epilepsy. Histological examination of brain sections showed that the rat model of PTE was successfully established. At the PTE foci, high α-helical absorption regions were evident, where the color difference and absorption were significantly different from those in the low-absorption regions. This provided a distinctive and characteristic pattern at the site of lesions. The use of FTIR imaging means that it is possible to measure the molecular structural changes resulting from PTE pathologies in tissues, providing a novel adjunct to conventional pathological diagnostic techniques.
... While the infrared and Raman spectral signatures of superficial, intermediate, and parabasal cell layers have previously been investigated in cervical tissue, 14,15,35,36 to the best of our knowledge, this has not previously been investigated in single exfoliated cervical cells. ...
It is widely accepted that cervical screening has significantly reduced the incidence of cervical cancer worldwide. The primary screening test for cervical cancer is the Papanicolaou (Pap) test, which has extremely variable specificity and sensitivity. There is an unmet clinical need for methods to aid clinicians in the early detection of cervical precancer. Raman spectroscopy is a label-free objective method that can provide a biochemical fingerprint of a given sample. Compared with studies on infrared spectroscopy, relatively few Raman spectroscopy studies have been carried out to date on cervical cytology. The aim of this study was to define the Raman spectral signatures of cervical exfoliated cells present in liquid-based cytology Pap test specimens and to compare the signature of high-grade dysplastic cells to each of the normal cell types. Raman spectra were recorded from single exfoliated cells and subjected to multivariate statistical analysis. The study demonstrated that Raman spectroscopy can identify biochemical signatures associated with the most common cell types seen in liquid-based cytology samples; superficial, intermediate, and parabasal cells. In addition, biochemical changes associated with high-grade dysplasia could be identified suggesting that Raman spectroscopy could be used to aid current cervical screening tests. © 2017 Society of Photo-Optical Instrumentation Engineers (SPIE).
... In the 1100–900 cm –1 spectral range, several macromolecules such as polysaccharides and phosphate carrying compounds (such as phospholipids and nucleic acids) give absorption bands [19, 22]. This region has been shown to be a major indicator of carcino- genesis [23][24][25]. Many peaks found in this region can be used to classify tumors. ...
This study proposes Fourier Transform Infrared (FTIR) spectroscopy as a more sensitive, rapid, non‐destructive and operator‐independent analytical diagnostic method for bladder cancer recurrence from bladder wash than other routinely used urine cytology and cystoscopy methods.
A total of 136 patients were recruited. FTIR spectroscopic experiments were carried out as a blind study, the classification results of which were then compared with those of cytology and cystoscopy. Firstly, 71 samples ( n = 37; bladder cancer and n = 34; control) were studied with transmittance FTIR spectroscopy. After achieving successful differentiation of the groups, to develop a more rapid diagnostic tool and check the reproducibility of the results, the work was continued with different samples ( n = 65 as n = 44; bladder cancer and n = 21; control), using the reflection mode (ATR) of FTIR spectroscopy by a different operator. The results revealed significant alterations in moleculer content in the cancer group. Based on the spectral differences, using transmittance FTIR spectroscopy coupled with chemometrics, the diseased group was successfully differentiated from the control. When only carcinoma group was taken into consideration a sensitivity value of 100% was achieved. Similar results were also obtained by ATR‐FTIR spectroscopy. This study shows the power of infrared spectroscopy in the diagnosis of bladder cancer. magnified image
... 99 These studies have included analysis of cell maturation in cervical tissue where changes in glycogen concentration could be associated with the different stages of cell maturation. 100, 101 Cohenford and coworkers have also undertaken several studies including analysis of cervical smears 102 and demonstrated that FT-IR, as well as aiding in the diagnosis of cervical neoplasia, could also provide information into its pathogenesis. 103 This same group also analysed FT-IR spectra of over 2000 individual cells. ...
Vibrational microspectroscopic (Raman and infrared (IR)) techniques are rapidly emerging as effective tools to probe the basic processes of life. This project mainly focuses on the applications of Raman and IR microspectroscopy to biology and biomedicine, ranging from studies on cellular components in single cells to advancement in techniques for in vitro to in vivo applications. These techniques have proved to be instrumental in studying the biological specimen with minimum perturbation, i.e. without the use of dyes and contrast inducing agents. These techniques probe the vibrational modes of the molecules and provide spectra thatare specific to the molecular properties and chemical nature of the species. The ability to diagnose the early onset of disease, rapidly, non-invasively and unequivocally has multiple benefits. These include the early intervention of therapeutic strategies leading to a reduction in morbidity and mortality, and the releasing of economic resources within overburdened health care systems. In addition, these often rely on single disease markers which are inappropriate when multiple factors are involved. Many diseases are a result of metabolic disorders, therefore it is logical to measure metabolism directly. One of the strategies employed by the emergent science of metabolomics is metabolic fingerprinting; which involves rapid, high-throughput global analysis to discriminate between samples of different biological status or origin. This project focuses on a selective number of recent studies where metabolic fingerprinting has been forwarded as a potential tool for disease diagnosis using infrared and Raman spectroscopies. Quantum computation in the brain, emergence of unperturbed mind and behavioural pattern due to resistive, inductive and capacitative bis loads.
... On the other hand, there is an alternative technique based on tissue level known as hyperspectral diagnostic imaging (HSDI). The surface of the cervix is scanned with ultraviolet and white light for detecting lesions [51]. The scanning is achieved one line at a time, with the scan time varying from 12 to 24 seconds. ...
Advancement of medical image digitalization leads to image processing and computer-aided diagnosis systems in numerous clinical applications. These technologies could be used to automatically diagnose patient or serve as second opinion to pathologists. This paper briefly reviews cervical screening techniques, available so that this can motivate the researcher to concentrate more on medical related issues which becomes a small contribution to our country which is very essential. The digital data of the screening techniques are used as data for the computer screening system as replaced in the expert analysis. Four stages of the computer system are enhancement, features extraction, feature selection, and classification reviewed in detail.
... This latter change is most likely due to variations in carbohydrate (glycogen) concentration, see Fig. 1. A decrease in the glycogen signal of diseased cells has also been reported previously in the analysis of cervical cells 39 and has been reported for Barrett's associated changes by Stone,et al. 40 An interesting observation is presented in Fig. 10a, namely the aforementioned appearance of the shoulder at 1635 cm −1 in the spectra of the squamous cells. This peak was previously observed in tissue that has pathologically identified necrotic regions; 25c and Jamin et al., 38 have associated the corres- ponding shoulder at the amide I band with apoptosis or necrosis. ...
Instrumental advances in infrared micro-spectroscopy have made possible the observation of individual human cells and even subcellular structures. The observed spectra represent a snapshot of the biochemical composition of a cell; this composition varies subtly but reproducibly with cellular effects such as progression through the cell cycle, cell maturation and differentiation, and disease. The aim of this summary is to provide a synopsis of the progress achieved in infrared spectral cytopathology (SCP) - the combination of infrared micro-spectroscopy and multivariate methods of analysis - for the detection of abnormalities in exfoliated human cells of the upper respiratory and digestive tract, namely the oral and nasopharyngeal cavities, and the esophagus.
... By using this parameter in conjunction with identified earlier changes in band intensity of glycogen and phosphate groups, two distinct clusters normal and cancerous cervical tissues were reported [93]. however, further studies have demonstrated that the observed spectral changes might not be strictly correlated with the presence of dysplastic cells, but can result from differentiation, maturation, a stage of the menstrual cycle, contamination of mucin, blood cells, and other factors [94][95][96][97][98]. Consequently, these changes can also cause non-specific depletion of glycogen and variation of phosphate group bands. ...
In this chapter, we describe biomedical applications of infrared microscopic imaging applied to human tissue sections. The central focus is human diseases including cervical cancer, neurodegenerative pathologies, and dysfunctions of cardiac and liver tissues. In addition, we briefly describe the fundamentals of FTIR imaging instrumentation along with spectral pre-processing and hyperspectral image reconstruction. The chapter concludes with a summary of what is required to take FTIR imaging technology into the clinical environment.
... In recent years, Fourier transform infrared (FTIR) spectroscopy has been increasingly applied to the study of biomedical conditions, as it can permit the chemical characterization of microscopic areas in a tissue sample [3][4][5][6]. There are two types of FTIR detection: FTIR mapping and FTIR imaging. ...
A comprehensive, coherent, and in depth presentation of the state of the art in fuzzy clustering.
Fuzzy clustering is now a mature and vibrant area of research with highly innovative advanced applications. Encapsulating this through presenting a careful selection of research contributions, this book addresses timely and relevant concepts and methods, whilst identifying major challenges and recent developments in the area. Split into five clear sections, Fundamentals, Visualization, Algorithms and Computational Aspects, Real-Time and Dynamic Clustering, and Applications and Case Studies, the book covers a wealth of novel, original and fully updated material, and in particular offers:
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This book will be of key interest to engineers associated with fuzzy control, bioinformatics, data mining, image processing, and pattern recognition, while computer engineers, students and researchers, in most engineering disciplines, will find this an invaluable resource and research tool.
... This decrease in glycogen tends to agree with known biochemical changes leading to tumor development such as increased energy consumption from cell division and has been reported previously for Barrett's associated changes by Stone et al. 30,31 The diminished glycogen signal of cancerous and diseased cells has also been reported previously in the analysis of cervical cells. 32 Unsupervised analysis of the spectral data demonstrates that there are interpretable and reproducible changes that occur between the spectra of normal and diseased samples. In order to achieve a classification supervised, or trained algorithms, are used to predict class membership for individual cells. ...
We report results from a study utilizing infrared spectral cytopathology (SCP) to detect abnormalities in exfoliated esophageal cells. SCP has been developed over the past decade as an ancillary tool to classical cytopathology. In SCP, the biochemical composition of individual cells is probed by collecting infrared absorption spectra from each individual, unstained cell, and correlating the observed spectral patterns, and the variations therein, with against classical diagnostic methods to obtain an objective, machine-based classification of cells. In the past, SCP has been applied to the analysis and classification of cells exfoliated from the cervix and the oral cavity. In these studies, it was established that SCP can distinguish normal and abnormal cell types. Furthermore, SCP can differentiate between truly normal cells, and cells with normal morphology from the vicinity of abnormalities. Thus, SCP may be a valuable tool for the screening of early stages of dysplasia and pre-cancer.
... (1) image results: fluorescent in situ hybridization (FISH) [6][7][8][9][10][11]; (2) spectra results: Raman spectroscopy [12,13], fluorescence spectroscopy [14,15], and Fourier transform infrared (FTIR) spectroscopy [16][17][18][19][20][21][22][23][24]. ...
Advent of medical image digitalization leads to image processing and computer-aided diagnosis systems in numerous clinical applications. These technologies could be used to automatically diagnose patient or serve as second opinion to pathologists. This paper briefly reviews cervical screening techniques, advantages, and disadvantages. The digital data of the screening techniques are used as data for the computer screening system as replaced in the expert analysis. Four stages of the computer system are enhancement, features extraction, feature selection, and classification reviewed in detail. The computer system based on cytology data and electromagnetic spectra data achieved better accuracy than other data.
... Several research groups reported its use for identification, differentiation, and classification of microorganisms (10, 11). Among its numerous applications, differentiation of normal from cancerous cervical tissue, normal and cancerous colon tissue, normal and malignant lymph cells and tissue, and investigation of brain cancer were reported (12–16). To improve FT-IR results, several studies have combined FT-IR results with chemometrics (17–21). ...
The seminal plasma is an excellent source for noninvasive detection of spermatogenesis. The seminal plasma of normospermic and azoospermic men has been analyzed for detection of spermatogenesis.
Optical spectroscopy (Attenuated Total Reflectance-Infrared spectroscopy (ATR-IR) and Fourier Transform infrared spectroscopy (FT-IR) has been used to analyze the seminal plasma and the metabolome of seminal plasma for detection of spermatogenesis.
The seminal plasma of normospermic and azoospermic men has been analyzed by ATR-IR. The results show that there is a pattern variation in the azoospermic men compared to normospermic men. However, the seminal plasma is too complex to show significant pattern variation. Therefore, the metabolome which is a subcomponent of the seminal plasma was analyzed. The seminal plasma metabolome of normospermic and azoospermic men has been analyzed by FT-IR. A significant pattern change was observed. The data combined with chemometrics analysis showed that significant changes are observed at metabolome level.
We suggest that FT-IR has the potential as a diagnostic tool instead of testicular biopsy.
... Since then most FTIR studies have been performed on exfoliated cervical cells and their main objective has been to improve the diagnostic accuracy of the Papanicolaou smear [163]. FTIR mapping [164,165] and FTIR imaging [10,166,167] examined cervical tissue sections. A villoglandular adenocarcinoma from a cervical biopsy was used as a model system to demonstrate a three-dimensional image processing technique for FTIR images [10]. ...
The objective of the article is to review biomedical applications which became possible after the development of sensitive and high throughput Raman and Fourier transform infrared spectrometers in the past decade. Technical aspects of the instrumentation are briefly described. Then the broad range of vibrational spectroscopic applications with the focus on imaging and fiber-optical methods are discussed to study mineralized tissue (bone, teeth), skin, brain, the gastrointestinal tract (mouth, pharynx, esophagus, colon), breast, arteries, cartilage, cervix uteri, the urinary tract (prostate, bladder), lung, ocular tissue, liver, heart and spleen. Experimental studies are summarized demonstrating the possibilities and prospects of these methods in various fields of biodiagnostics to detect and characterize diseases, tumors and other pathologies.
... On the other hand, FTIR spectroscopy has been utilized as an emerging method applied to the study of the structural changes of cells at the molecular level in various human cancers . Over the past decades, there have been a number of studies done to investigate the possibility of the FTIR technique as a screening tool for cervical cancer161718192021. Inspired by the outstanding results, many researchers have worked to explore and apply the FTIR spectroscopy as a diagnostic tool for differentiating normal and malignant tissues and cells of several human cancers; including esophagus [22], colon [23], [24], skin [25], gastric [26], lung [27], gliomas [28,29], and cervical cancers [4,5,3031323334. ...
Inspired from the great potential of the Fourier-transform infrared (FTIR) spectroscopy as a screening tool for cervical cancer, this paper proposes an intelligent classification of cervical pre-cancerous cells based on the FTIR spectra. It consists of two parts: the extraction of FTIR characteristics and the intelligent classification of the pre-cancerous cells. Peak-corrected area-based features’ extraction (PCABFE) is introduced as a tool for the first part, while the Hybrid Multilayered Perceptron (HMLP) network is employed to classify the cervical pre-cancerous cells according to the Bethesda classification. Correlation test proves the capability of the proposed PCABFE to be as effective as the manual extraction by human experts, while the HMLP network produces a good classification performance with 97.4% of accuracy.
... Over the past decade, several research groups have used infrared spectroscopy and infrared microspectroscopy (IR-MSP) as a diagnostic tool to distinguish normal from diseased cells and tissue [1][2][3]. This distinction works on the principle that infrared spectroscopy monitors chemical composition, and changes therein, of a sample. ...
Infrared microspectroscopy (IR-MSP) is a spectroscopic technique that is able to monitor cell differentiation, maturation, and progression through the cell cycle. In order to establish this technique as a diagnostic tool in cellular biology and pharmacology, spectral patterns indicative of the stages of cell proliferation need to be collected. Thus, we have embarked on a systematic study of the effects of cell division and cell cycle progression on the infrared spectra of cells.In this paper, we modulated the level of cell proliferation and report the effects of this modulation on the observed infrared spectra of the cells. The modulation was achieved by serum deprivation of the growing cells, or by having the cell culture reach confluence. The progression of the cells through the cell cycle was monitored via flow cytometry, and correlated with changes in IR-MSP features in the spectral signatures due to nucleic acids (1250–1000 cm−1).In both these experiments, the majority of cells are in the G0/G1 stages,3 with only a small percentage in the S and G2 phases. Nevertheless, spectral differences could be observed and interpreted in terms of the spectral changes of cellular DNA.
... The distribution of normal, superficial, intermediate, parabasal, and basal cells can greatly influence not only the spectral collection from a Pap but also the Pap smear interpretation itself as shown by Bibbo et al. [5,6]. It has also been shown that benign, immature epithelial cells can closely approximate the infrared spectrum of abnormal and neoplastic cells [7]. In fact, recent evidence strongly suggests that the spectral differences between neoplastic and benign cells are quite small and may be difficult to resolve [8,9]. ...
... Since infrared spectroscopic methods are sensitive to the chemical/biochemical composition of histological samples, changes related to diseases may be observed as small variations in structural composition of cells and tissues. These changes may be translated into false color images by a number of chemometric methods that can be interpreted by non-spectroscopists, and are directly comparable to outcomes of standard histological staining protocols 3,4 . The paper we will discuss strategies of IR spectroscopic imaging of experimental data obtained on histological thin sections from the human prostate. ...
Different cluster image reassembling methodologies have been used to generate infrared maps from FT-IR microspectra of human prostate tissue sections. Spectra were collected in transmission mode with high spatial resolution by the use of a HgCdTe focal plane array detector imaging system. While univariate imaging techniques such as chemical mapping often give unsatisfactory classification results, unsupervised multivariate data analysis techniques such as agglomerative hierarchical clustering, fuzzy C-means, or k-means clustering confirmed standard histopathological techniques and turned out to be helpful to identify and to discriminate tissues structures. The use of any of the clustering algorithms dramatically increased the information content of the IR images, as compared to chemical mapping. Among the cluster imaging methods, agglomerative hierarchical clustering (Ward's algorithm) turned out to be the best method in terms of tissue structure differentiation.
... The authors reported a decrease in the intensity of glycogen bands and an increase in the intensity of symmetric and anti-symmetric phosphate (PO2 -) bands associated with nucleic acids for dysplastic and cancerous samples. However, further studies undertaken by Cohenford et al. [20,21], McNaugthton et al. [22][23][24] and Diem et al. [25][26][27][28][29] indicated that these spectral changes observed by Wong may not be related to the number or molecular composition of dysplastic cells, but to confounding contributions made by different cell types present within a smear. Benign variations such as inflammation, metaplasia, the ratio of non-dividing to dividing cells, and the overall divisional activity of the cells can dramatically change the IR spectrum collected. ...
... In the past decade Fourier transform infrared (FT-IR ) spectroscopy has become a useful analytical tool in biomedical science, e.g., for the characterization of microorganisms, 1 isolated cells or cell lines, 2-4 body uids, 5 and tissues, [6][7][8][9][10][11][12] to mention a few applications. FT-IR microspectroscopy, as the combination of FT-IR spectroscopy and m icroscopy, is proving to be an attractive new optical technique that provides objective structural information on minute microscopic structures. ...
A combination of Fourier transform infrared (FT-IR) spectroscopy and microscopy, FT-IR microspectroscopy, has been used to characterize sections of human colorectal adenocarcinoma. In this report, a database of 2601 high quality FT-IR point spectra from 26 patient samples and seven different histological structures was recorded and analyzed. The computer-based analysis of the IR spectra was carried out in four steps: (1) an initial test for spectral quality, (2) data pre-processing, (3) data reduction and feature selection, and (4) classification of the tissue spectra by multivariate pattern recognition techniques such as hierarchical clustering and artificial neural network analysis. Furthermore, an example of how spectral databases can be utilized to reassemble false color images of tissue samples is presented. The overall classification accuracy attained by optimized artificial neural networks reached 95%, highlighting the great potential of FT-IR microspectroscopy as a potentially valuable, reagent-free technique for the characterization of tissue specimens. However, technical improvements and the compilation of validated spectral databases are essential prerequisites to make the infrared technique applicable to routine and experimental clinical analysis.
... Fourier transform infrared (FT-IR) microspectroscopy of cells and tissues provides an immense amount of structural information. In the past 5 years, a number of research groups started a series of projects and tested the applicability of the spectroscopic technique to characterize tissue specimens [1][2][3][4][5]. These tests showed impressively that infrared signatures are potentially suitable for characterization of the samples in terms of histology and pathology if the infrared spectra were collected spatially resolved, i.e. by the use of an infrared microscope. ...
Spatially resolved Fourier transform infrared (FT-IR) spectroscopy of single oral mucosa cells and FT-IR spectroscopy of liver cell fractions produced by sucrose density gradient centrifugation have been applied to acquire structural information of cell organelles. For the spatially resolved measurements, mapping as well as mercury cadmium telluride (MCT) focal plane array (FPA) detector techniques were utilized. Surprisingly, infrared spectra of distinct subcellular structures differed only slightly. Aside from a minor intensity increase of the symmetric and the antisymmetric PO2− bands in the spectra of the nucleus, all normalized infrared spectra exhibited a remarkably high degree of similarity. Considering the fact that more than 98% of the DNA and significant amounts of the RNA are concentrated in the nucleus which occupies in oral mucosa cells only 5% of the total cell volume, these findings may be interpreted as a proof of the hypothesis that DNA is partially “invisible” in infrared spectroscopy.The finding of comparably small IR spectroscopic differences between cell organelles was confirmed by measurements on cell fractions specifically enriched by sucrose density gradient centrifugation. Besides variations of the non-specific overall protein:lipid ratio, the IR spectra of the distinct pellets exhibited only slightly differing absorbance values for the PO2− bands. As found for the nuclei by spatially resolved microspectrometry, IR spectra of the nuclear pellet displayed minor increased absorbances of the PO2− band at 1083cm−1 when compared to spectra of the mitochondrial or microsomal pellets.
... During the last decade, Raman spectroscopy and FT- IR micro-spectroscopy has already increased in capabilities to visual a molecular component for unstained tissue. 1 IR spectroscopy, which has low energy photons, are used to excite the vibrational motion of covalently bonded moieties by direct absorption of the photon. 2 Therefore, both of them has already become a powerful method for studying and investigating the complex molecular structures of samples. 1 We concerned and focused to phosphate caused of the phosphate stretching modes originate from the phosphodiester groups on nucleic acids, suggested an increase the nucleic acids in the malignant tissue. 3 Due to our goal to explore the intact information of human lung cancer tissue, which is not able to be carried out using H-E staining and also particuarly to distinct the grade within the cancer, we evaluated our studying with non pretreatment of sample tissue using FT-IR 3000M. ...
Ball-lens hollow Raman Probe (BHRP) and FTIR spectroscopy were used in this study. BHRP was purposed to explore the biochemical alteration in the living animal (in vivo study), mean while FTIR spectroscopy was introduced for clarification of BHRP result. In this study, we focused to the alteration of antisymmetric and symmetric P=O stretching vibration within the azoxymethane-induced colorectal tumor (in mouse). BHRP detected some differences of it due to randomly the edge of BHRP attached the surface of tumor. Meanwhile, the application of FTIR were introduced to differentiate between grade levels of non clinic samples colorectal tumor models at 4 different grades; normal, grade 1, grade2 and grade 3. Detailed investigation of the spectra in the fingerprint region 1800-700 cm -1 for BHRP and 4000–500 cm -1 for FTIR revealed some distinct peaks and shoulders, most of which were assignable to wave numbers that shown to represent biochemical changing within the tissue. Differences in peak heights and peak ratios indicated differences in biochemical composition of cancer from different grade level. It was possible to distinguish between their grade. However, all collected colorectal tumor model at different peak was distinguishable, where antisymmetric stretching of CH 2 , antisymmetric and symmetric P=O stretching vibration was imaged and mapped clearly. Therefore, BHRP collaborated with FTIR spectral analysis and in combination with calibration curve might be used to distinguish cancer grade within colorectal tumor model tissue. Extension of this method to other cancer type may be of great interest for cancer grade determination.
Infrared (IR) spectroscopic imaging records spatially resolved molecular vibrational spectra, enabling a comprehensive measurement of the chemical makeup and heterogeneity of biological tissues. Combining this novel contrast mechanism in microscopy with the use of artificial intelligence can transform the practice of histopathology, which currently relies largely on human examination of morphologic patterns within stained tissue. First, this review summarizes IR imaging instrumentation especially suited to histopathology, analyses of its performance, and major trends. Second, an overview of data processing methods and application of machine learning is given, with an emphasis on the emerging use of deep learning. Third, a discussion on workflows in pathology is provided, with four categories proposed based on the complexity of methods and the analytical performance needed. Last, a set of guidelines, termed experimental and analytical specifications for spectroscopic imaging in histopathology, are proposed to help standardize the diversity of approaches in this emerging area.
Expected final online publication date for the Annual Review of Analytical Chemistry, Volume 16 is June 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Fourier-transform infrared (FTIR) microscopy is considered a comprehensive and sensitive method for detection of molecular changes in cells. The advantage of FTIR microspectroscopy over conventional FTIR spectroscopy is that it facilitates inspection of restricted regions of a cell culture or a tissue. We have shown that it is possible to apply FTIR microscopy as a sensitive and effective assay for the detection of cells infected with various members of the herpes family of viruses and retroviruses. Detectable and significant spectral differences between normal and infected cells were evident at early stages of the infection. Impressive changes in several spectroscopic parameters were seen in infected compared with uninfected cells. It seems that the change in spectral behavior is specific to the infecting virus, because cells infected with herpesviruses showed different spectral changes compared with cells infected with retoviruses.
Spectroscopic techniques have been finding increasing applications in the field of biomedicine especially in the field of disease diagnosis and monitoring in spite of the rapid emergence of several molecular biology based techniques. The significance of spectroscopy techniques and the possibility of using some of the underutilized regions of the electromagnetic radiations are discussed in this review. While previous reviews have already dealt with the potential of Fourier transform infrared spectroscopy-based (FTIR) techniques for clinical applications, the present review addresses the lacunae of the techniques along with its future trends that may make it a technique routinely applied in clinical settings.
Fourier transform infrared (FT-IR) spectroscopy is a rapid, reagent-less, non-destructive, analytical technique whose continuing development is resulting in manifold applications in the biosciences. The principle of FT-IR lies in the fact that when a sample is interrogated with light (or electromagnetic radiation), chemical bonds absorb at specific wavelengths and vibrate in one of a number of ways. These absorptions/vibrations can then be correlated to the bonds or functional groups of molecules. Because of its chemical information content and spectral richness (defined as numbers of clearly defined peaks) the major wavenumber region of interest is the mid-infrared, usually defined as 4000–600 cm-1 (see Table 1). The infrared spectra of proteins, as a prominent example, exhibit strong amide I absorption bands at 1653 cm-1 associated with characteristic stretching of C=O and C-N and bending of the N-H bond (Stuart, 1997).
This comprehensive overview of biomedical applications of vibrational spectroscopy focuses on methodologies that are most relevant to biodiagnostics. After a few introductory chapters that summarize the current status of the field, the reference covers current spectroscopic applications; new spectroscopic directions; and study design and the analysis of vibrational spectral fingerprints from complex biological and clinical samples . With chapters contributed by leading international experts, Biomedical Vibrational Spectroscopy is a core resource
Background: The seminal plasma is an excellent source for noninvasive detection
of spermatogenesis. The seminal plasma of normospermic and azoospermic
men has been analyzed for detection of spermatogenesis.
Methods: Optical spectroscopy (Attenuated Total Reflectance-Infrared spectroscopy
(ATR-IR) and Fourier Transform infrared spectroscopy (FT-IR) has
been used to analyze the seminal plasma and the metabolome of seminal
plasma for detection of spermatogenesis.
Results The seminal plasma of normospermic and azoospermic men has been
analyzed by ATR-IR. The results show that there is a pattern variation in the
azoospermic men compared to normospermic men. However, the seminal
plasma is too complex to show significant pattern variation. Therefore, the
metabolome which is a subcomponent of the seminal plasma was analyzed.
The seminal plasma metabolome of normospermic and azoospermic men has
been analyzed by FT-IR. A significant pattern change was observed. The data
combined with chemometrics analysis showed that significant changes are observed
at metabolome level.
Conclusion: We suggest that FT-IR has the potential as a diagnostic tool instead
of testicular biopsy.
Recent papers comparing the relative signal to noise performance of filters and interferometers for visible and near-IR spectral imaging have reached divergent conclusions. Others have presented the proposition that interferometer systems, by capturing light from multiple spectral channels bands at once, inherently outperform filter-type systems, which capture a single channel at a time. In this paper, a general analysis is provided that establishes a basis for comparison between band-sequential spectrometers and imaging interferometers in the shot-noise limit (set by Poisson statistics of the number of electrons produced at the detector), and in practice. It is shown that factors such as lamp flicker and sample stability can introduce much more error than shot noise does. This, one must be aware that the use of shot-noise limited detectors does not ensure shot- noise limited images.
Indocyanide green (ICG) is widely used as a tracer for the non-invasive estimation of liver function. ICG has properties of binding with plasma protein, and has a large absorption peak at 805 nm. There were no reports, however, about the IR absorption peak of ICG at 7.1 micrometers , which absorption coefficient amounts to approximately 13000cm-1. In this study, ICG was exposed to free electron lasers (FELs) with wavelength of 7.1 micrometers and usefulness of ICG as an IR-marker was discussed. ICG film sample was formed on IR-transparent BaF2 crystal substrate and exposed to FELs with the wavelength of 7.1micrometers . After exposure the sample was analyzed by FT-IR and film thickness measurements. As results, ICG ablated with the FEL of the power density of more than 5 W/cm2(equalsPdth), and that the molecular structure of ICG was still stable for the power density of less than Pdth, 3 W/cm2. Therefore, ICG can be considered as a novel infrared marker (IR marker) to the living tissue which absorbs FEL photon energy without changing the IR absorption peak.
As a precursor to applying fluorescence lifetime imaging (FLIM) to studies of intercellular communication in molecular immunology, we have investigated the fluorescence lifetime of enhanced green fluorescent protein (EGFP) in mixtures of water and glycerol using time-correlated single photon counting (TCSPC). We find that the EGFP lifetime decreases with increasing glycerol content. This is accounted for quantitatively by the refractive index dependence of the fluorescence lifetime as predicted by the Strickler Berg formula which relates the fluorescence lifetime to the absorption spectrum. The solvent viscosity has no influence on the fluorescence lifetime. We also discuss the refractive index dependence of the GFP fluorescence lifetime in more complex systems. The findings are particularly relevant for the interpretation of FLIM of GFP expressed in environments such as bacteria and cells.
Synchrotron FTIR maps, focal plane array and linear array images recorded of 4 mum cervical biopsy sections from the surface epithelium and glandular endometrium are compared in terms of spatial resolution and applicability to the clinical environment. Synchrotron FTIR maps using a 10 mum aperture appear to provide a better spatial resolution capable of discerning single nuclei in the tissue matrix. Unsupervised hierarchical cluster analysis performed on the synchrotron, focal plane array and linear array data in the 1700-1400 cm-1 region show very similar clusters and mean-extracted spectra, demonstrating the robustness of FTIR microscopy and UHCA in the analysis of tissue sections. Maps recorded with the focal plane array using a conventional globar source take one-fortieth of the time but the spatial resolution precludes true single cell analysis in the tissue matrix. The high spatial resolution achieved with the synchrotron shows potential as a gold standard for FTIR diagnosis of cervical samples.
Oral cancer and precancer overexpress the epidermal growth factor receptor (EGFR) and monoclonal antibodies against EGFR coupled to photoactive dyes may have a potential both as a diagnostic and treatment modalities for oral premalignancy. We asked whether an anti-EGFR mab (C225) conjugated with the fluorescence dye indocyanine Cy5.5 could detect dysplastic changes in the hamster cheek pouch carcinogenesis model. Secondly, we tested whether the same antibody conjugated with the photosensitizer chlorin (e6) could be used together with illumination to reduce levels of expression of EGFR as evaluated by the immunophotodetection procedure. Increased fluorescence appeared to correlate with development of premalignancy when the C225-Cy5.5 conjugate was used. Areas with increased fluorescence signal were found in carcinogen-treated but clinically normal cheek pouches, that revealed dysplastsic changes by histology. The immunophotodetection procedure was carried out after photoummunotherapy with the C225-ce6 conjugate, and showed a significant reduction in fluorescence in the illuminated compared to the non-illuminated areas in the carcinogen- treated but not the normal cheek pouch. The results demonstrate that the use of anti-EGFR Mab targeted photoactive dyes may serve as a feedback controlled optical diagnosis and therapy procedure for oral premalignant lesions.
We have applied Fourier transform infrared (FTIR) spectroscopic imaging, coupling a mercury cadmium telluride (MCT) focal plane array detector (FPA) and a Michelson step scan interferometer, to the investigation of various states of malignant human prostate tissue. The MCT FPA used consists of 64x64 pixels, each 61 micrometers 2, and has a spectral range of 2-10.5 microns. Each imaging data set was collected at 16-1 resolution, resulting in 512 image planes and a total of 4096 interferograms. In this article we describe a method for separating different tissue types contained within FTIR spectroscopic imaging data sets of human prostate tissue biopsies. We present images, generated by the Fuzzy C-Means clustering algorithm, which demonstrate the successful partitioning of distinct tissue type domains. Additionally, analysis of differences in the centroid spectra corresponding to different tissue types provides an insight into their biochemical composition. Lastly, we demonstrate the ability to partition tissue type regions in a different data set using centroid spectra calculated from the original data set. This has implications for the use of the Fuzzy C-Means algorithm as an automated technique for the separation and examination of tissue domains in biopsy samples.
This proceedings contains papers on the following topics: spectral
imaging data, multivariate data processing exogenous optical contrast
agents, imaging of ions and metabolites by fluorescence microscopy.
The human eyes are not made to detect disease, however visual perception is the most common screening method for early cancer detection. With optimal illumination and observation configuration there is significant improvement of optical contrast between normal and pre-cancerous tissue in the oral cavity, both for reflected and fluorescent light.
Five peptides binding to somatostatin and bombesin receptors were conjugated to 4-azido-2,3,4,6-tetrafluorophenylbenzoic acid, a Type 1 photosensitizer, at the N-terminal position. The receptor affinities were determined by competition binding assay using two different pancreatic tumor cell lines, CA20948 and AR42-J, that expresses somatostatin-2 (SST-2) and bombesin receptors receptively. All compounds exhibited high receptor specificity, i.e., the IC50 values ranged between 1.0 to 64.0 nM. These conjugates may be useful for targeted Type 1 phototherapy via the generation of nitrenes at the cell surfaces expressing these receptors.
Fourier transform infrared (FTIR) spectroscopy was explored as a means to distinguish newly diagnosed of acute lymphoblastic leukemia from disease free bone marrow samples. Characteristic bands alterations were identified in both healthy and diseased samples arising from cellular protein, lipid and DNA. There were specific changes that affected the secondary structure of proteins that appeared in the FTIR spectra and confirmed with the second derivative analysis. The overall protein structure in the control sample consists primarily of α-helix, whereas in ALL sample it has a relatively high proportion of anti-parallel β-sheet protein constituents presumably due to leukemia. Different absorbance's ratios for specific bands were calculated and plotted versus the patient samples. There are significant fluctuations in the ratios under investigation which can be attributed to the changes in the biomolecular structure between normal and leukemia samples. Our results indicate that the absorbance of amide A and B are in the range 3,340-3,000, the lipid/protein ratio and the phosphate/amide II ratio are all yielding statistically significant differences parameters, that it can be used as a biomarker in differentiating acute leukemia from leukemia free bone marrow.
The spectroscopy of molecular vibrations using mid-infrared or Raman techniques has been applied to samples of human tissue or body fluids. This review gives examples for its potential in supporting medical diagnostics. Most of the investigations have been performed in the field of internal medicine, namely angiology, hematology, rheumatology, endocrinology, gastroenterology, and nephrology. Further potential applications in neurology, gynecology, obstetrics and dermatology are considered. Although some fundamental issues of the vibrational spectroscopy of biomedical samples still have to be solved, significant progress has been achieved in its clinical application in-vitro. First steps towards in-vivo applications are described.
Cancer of uterine cervix is one of the leading cancers among women in both developed and developing countries. Optical spectroscopy methods mostly Fourier transform infrared (FTIR) and fluorescence have widely been used to diagnose cervix cancer using cells as well as tissues. Raman spectra of normal and malignant tissues were recorded in fingerprint region. Normal cervix tissues are characterized by strong, broad amide I, broader amide III and strong peaks at 853 and 938cm−1 which can be attributed to structural proteins such as collagen. Prominent features of malignant tissue spectra with respect to normal tissue are—relatively weaker and sharper amide I, minor red shift in ΔCH2 and sharper amide III which indicate the presence of Deoxyribonucleic acid (DNA), lipids and non-collagenous proteins. In order to develop highly objective discrimination methods, very elaborate data analysis was carried out using Principal Components Analysis (PCA). Standard sets for normal and malignant were prepared and tested retrospectively and prospectively. Several parameters such as (scores of factor, Mahalanobis distance and spectral residuals) were explored for discrimination and very clean clustering of normal and malignant spectra was achieved. A multiparametric approach (limit test) combining all the above discrimination parameters was also considered, in order to develop unambiguous discrimination. This analysis has produced very high, 99.5%, sensitivity and specificity. Results obtained in this study thus validate Raman spectroscopy methods for discrimination of normal and malignant tissues in cervical cancers.
FT-IR microimaging was performed on colon tissues with the aim to characterize spectral ‘markers’ to distinguish healthy from pathological tissues. Evidences of spectral peculiarities were mainly found in the finger print region even in the presence of a low grade adenocarcinoma. The occurrence of inflammation and necrotic states can also be demonstrated. Through statistical analysis as well as custom map procedures it was possible to reconstruct the topological distribution of different biochemical states and to verify results from the histopathological analysis. Preliminary results from FT-NIR analysis are in substantial agreement with those in the mid infrared region.
Ball-lens hollow fiber Raman Probe (BHRP) and FTIR spectroscopy were main tools in this study. Thus, both of equipments detected the alteration of antisymmetric and symmetric P=O stretching vibration within our mice colorectal tumor models. Some differences of spectra due to randomly the edge of each BHRP and FTIR attached the surface of tumor during measurements. Meanwhile, the application of FTIR potentially differentiates the grade levels of non-clinic samples colorectal tumor models at four different grades (normal, grade 1, grade 2 and grade 3). Detailed investigations were assignable to wave numbers that publicized to represent biochemical alteration. The whole of investigated spectra in the fingerprint region revealed some different peaks and shoulders, most of which were assignable to wave numbers that exposed to represent biochemical alteration within the tissue. Differences in peak heights and peak ratio indicated differences in biochemical composition of cancer from different grade level. However, all collected colorectal tumor model at different peak was distinguishable, where antisymmetric and symmetric P=O stretching vibration was imaged and mapped clearly by both equipments. Therefore, BHRP were comfortable for in vivo studies. Meanwhile FTIR spectral analysis in combination with calibration curve might be used to distinguish cancer grade within colorectal tumor model tissue for ex vivo study.
From November 1, 1997 till November 1, 2000 we have investigated 204 cases of acute myeloid leukemia (AML) (nequals95), acute lymphatic leukemia (ALL) (nequals40), myelodysplastic syndrome (MDS) (nequals11), chronic myeloid leukemia (CML) (nequals9), chronic lymphatic leukemia (CLL) (nequals4) and non-Hodgkin lymphoma (NHL) (nequals45) cytogenetically, using G-band analysis and spectral karyotyping (SKY). By SKY we were able to detect the abnormal clones in all cases but 9. In the G-band preparations these cases showed very few abnormal mitoses. The SKY either extended or confirmed the G-band findings in 94% of those with an abnormal karyotype. Cryptic translocations (translocations not suspected from the G-band karyotype) were found in 71 cases (26 AML, 9 ALL, 5 MDS, 2 CLL and 29 NHL). We find SKY a powerful adjuvant diagnostic tool that does not compromise one of the advantages of karyotyping techniques, the analysis of the entire genome which, in contrast to molecular biological techniques, still leave the possibility to get mroe answers than questions posed.
The Optical Sciences & Engineering Research Center (OSER) at Virginia Polytechnic and State University investigates advanced laser surgery optics, biocompatible material for implants, and diagnostic patches and other diagnostic and drug delivery tools. The Center employs optics to provide new biological research tools for visualization, measurement, analysis and manipulation. The Center's Research into Multispectral Medical Analysis and Visualization techniques will allow human and veterinary medical professionals to diagnose various conditions of the body in much the same way that satellite information is used to study earth resources. Each pixel in the image has an associated spectra. Advanced image analysis techniques are combined with cross-correlation of the spectra with signatures of known conditions, allowing automated diagnostic assistance to physicians. The analysis and visualization system consists of five components: data acquisition, data storage, data standardization, data analysis, and data visualization. OSER research efforts will be directed toward investigations of these system components as an integrated tool for next generation medical diagnostics. OSER will research critical data quality and data storage issues, mult-spectral sensor technologies, data analysis techniques, and diagnostic visualization systems including the VT-CAVE, (www.cave.vt.edu). The VT-CAVE is Virginia Tech's configuration of Fakespace Systems, Inc Virtual Reality system.
A high-performance hyperspectral imaging module with high light throughput suitable for microscopy and analytical imaging was built and tested. The imager utilizes the phenomenon of optical activity. The new technique provides information from a continuous spectral range of 250 - 1000nm. Similar spectral range extended to the near IR is also achievable. The imager has the form of a small module which can be inserted between a microscope or other imaging system and a camera. We have tested an 8-bit CCD video-rate camera with satisfactory results. The resulting instrument is simple, robust, and highly compact. The imager module is placed in-line to the microscope imaging system and does not introduce observable image aberrations. The imager is transparent to conventional imaging operations, thus with the imager in-place there is no need for reconfiguration of the microscope or switching between conventional and hyperspectral video/digital imaging modes. The presented spectral imager answers the need for a sensitive, compact, and affordable imaging spectrometer. The instrument is suited for applications requiring parallel acquisition of highly resolved concurrent spatial and spectral information such as high throughput screening, biochip analysis, remote sensing, semiconductor testing, etc. Images, spectral maps, and spectra of various fluorescent objects are presented.
Previous Fourier transform infrared (FT-IR) spectroscopic studies on neoplastic and normal cells have shown different band profiles and intensity associated with absorptions of proteins and nucleic acids. In the present study, an interpretation of such differences has been attempted by comparing the spectra of DNA/RNA/protein mixtures with the spectra, particularly, obtained for lymphocytes from B-chronic lymphatic leukemia (B-CLL) patients and normal donors. FT-IR microspectroscopy analysis showed a good agreement between the intensity and the band profile of the spectra of leukemic lymphocytes and those of the binary mixture made up of 75% human serum albumin and 25% DNA. The addition of small amounts of RNA (1-5% ) modified the band shape, making it more similar to the spectrum of normal lymphocytes. An attempt was also made to estimate the relative amounts of DNA and RNA. The results demonstrated an increase in the DNA/RNA ratio value in neoplastic lymphocytes with respect to that reported in literature for normal ones.
Infrared spectra were obtained from exfoliated cervical cells from 156 females, of whom 136 were normal, 12 had cervical cancer, and 8 had dysplasia. The spectra of the normal women, essentially identical, differed from those obtained from patients with either cancer or dysplasia. In malignant samples we noted (i) significant changes in the intensity of the glycogen bands at 1025 cm-1 and 1047 cm-1, the bands at 1082 cm-1 and 1244 cm-1, the C--O stretching band at 1155 cm-1, and the band at 1303 cm-1, (ii) significant shifts of the peaks normally appearing at 1082 cm-1, 1155 cm-1, and 1244 cm-1, and (iii) an additional band at 970 cm-1. Further study of several of these bands, including the pressure dependence of their frequencies, revealed that in the malignant cervical tissue there were extensive changes in the degree of hydrogen bonding of phosphodiester groups of nucleic acids and C--OH groups of proteins, as well as changes in the degree of disorder of methylene chains of lipids. The IR spectra of samples with dysplasia demonstrated the same changes with cancer samples, except that the changes were of lesser magnitude and the phosphodiester peak normally appearing at 1082 cm-1 did not shift. These spectroscopic changes appear to progress in tandem with the morphological changes that lead normal cervical epithelium to cancer through the premalignant stage of dysplasia. The diagnostic potential of IR spectroscopy is discussed.
Infrared microspectroscopy of biopsied canine lymph cells and tissue was performed to investigate the possibility of using IR spectra coupled with multivariate classification methods to classify the samples as normal, hyperplastic, or neoplastic (malignant). IR spectra were obtained in transmission mode through BaF2 windows and in reflection mode from samples prepared on gold-coated microscope slides. Cytology and histopathology samples were prepared by a variety of methods to identify the optimal methods of sample preparation. Cytospinning procedures that yielded a monolayer of cells on the BaF2 windows produced a limited set of IR transmission spectra. These transmission spectra were converted to absorbance and formed the basis for a classification rule that yielded 100% correct classification in a cross-validated context. Classifications of normal, hyperplastic, and neoplastic cell sample spectra were achieved by using both partial least-squares (PLS) and principal component regression (PCR) classification methods. Linear discriminant analysis applied to principal components obtained from the spectral data yielded a small number of misclassifications. PLS weight loading vectors yield valuable qualitative insight into the molecular changes that are responsible for the success of the infrared classification. These successful classification results show promise for assisting pathologists in the diagnosis of cell types and offer future potential for in vivo IR detection of some types of cancer.
Fourier-transform infrared (FTIR) microspectroscopy, combined with Principal Component Analysis (PCA), was applied in the study of exfoliated cervical cells from 272 patients. Six spectra were recorded for each patient, and these were visually sorted into two types (type 1 and type 2), based on their profiles. Spectra designated type 1 exhibited a profile characteristic of normal epithelial cells, with intense glycogen bands at 1022 cm−1 and 1150 cm−1, and a pronounced symmetric phosphate stretch at 1078 cm−1. Spectra designated type 2 exhibited features suggestive of dysplastic or malignant transformation, with pronounced symmetric and asymmetric phosphate modes and a reduction in glycogen-band intensity.Of the 272 patients, 68.6% of samples exhibited only type 1 profiles for all six recorded spectra, 29.4% of samples yielded at least one type 2 spectrum in any of the six recorded spectra and 2% of samples were inconclusive. Of the 68.6%, 86% were diagnosed normal by Pap smear with no follow up biopsy ordered, 7% were diagnosed abnormal by biopsy, 5% normal by biopsy and 2% were still inconclusive. For the remaining 29.4% of classified samples, 71% had shown an abnormal Pap result. These 71% were subsequently biopsied, and 87% were confirmed abnormal. The association of type 2 spectra and abnormality was further corroborated by spectra of cultured malignant cells from the HeLa cell line that displayed a profile similar to type 2 spectra in the 1300-950 cm−1 region. PCA decomposition using a reduced data matrix resulted in a score plot that showed general separation of the visually categorised spectra. This study demonstrates the potential of automated FTIR cervical screening technology in the clinical environment. © 1996 John Wiley & Sons, Inc.
Fourier transform infrared spectroscopy has been applied to the study of human breast tumors, human breast tumor cell lines and xenografted human tumor cells. The results presented indicate that substantial differences exist on a macroscopic level between human tumors, xenografted tumors and human tumor cell lines, which are related to the presence of a significant connective tissue matrix in the tumors. On a macroscopic level tumor cell xenografts appear, in spectroscopic terms, to be relatively homogeneous with a relatively weak signature characteristic of connective tissue. Differences on a microscopic level between adjacent small (30 mu m(2)) areas of the same xenografted tumor could be detected, which were due to local variations in collagen content. In addition to variations in collagen content, variation in the deposition of microscopic fat droplets throughout both human and xenografted tumors could be detected. These results indicate the care with which infrared spectroscopic studies of tissues must be carried out to avoid incorrect interpretation of results due to an incomplete understanding of tissue pathology. (C) 1995 John Wiley & Sons, Inc.