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Evaluating Different Fixation Protocols for Spectral Cytopathology, Part 2: Cultured Cells
Abstract
Spectral cytopathology (SCP) is a robust and reproducible diagnostic technique that employs infrared spectroscopy and multivariate statistical methods, such as principal component analysis to interrogate unstained cellular samples and discriminate changes on the biochemical level. In the past decade, SCP has taken considerable strides in its application for disease diagnosis. Cultured cell lines have proven to be useful model systems to provide detailed biological information to this field; however, the effects of sample fixation and storage of cultured cells are still not entirely understood in SCP. Conventional cytopathology utilizes fixation and staining methods that have been established and widely accepted for nearly a century and are focused on maintaining the morphology of a cell. Conversely, SCP practices must implement fixation protocols that preserve the sample's biochemical composition and maintain its spectral integrity so not to introduce spectral changes that may mask variance significant to disease. It is not only necessary to evaluate the effects on fixed exfoliated cells but also fixed cultured cells because although they are similar systems, they exhibit distinct differences. We report efforts to study the effects of fixation methodologies commonly used in traditional cytopathology and SCP including both fixed and unfixed routines applied to cultured HeLa cells, an adherent cervical cancer cell line. Data suggest parallel results to findings in Part 1 of this series for exfoliated cells, where the exposure time in fixative and duration of sample storage via desiccation contribute to minor spectral changes only. The results presented here reinforce observations from Part 1 indicating that changes induced by disease are much greater than changes observed as a result of alternate fixation methodologies. Principal component analysis of HeLa cells fixed via the same conditions and protocols as exfoliated cells (Part 1) yield nearly identical results. More importantly, the overall conclusion is that it is necessary that all samples subjected to comparative analysis should be prepared identically because although changes are minute, they are present. F or the past decade, infrared (IR) microspectroscopy has climbed its way to being considered a competitive alternative to conventional cytopathology practices. Traditional cytopathology includes the inspection of stained cells, visually measuring predetermined parameters, such as nucleus-to-cytoplasm (N/C) ratio, staining patterns, morphology of nuclear membrane, etc., and assigning a diagnosis based on these parameters. 1,2 IR microspectroscopy is at the forefront of new methods being developed because it is a label free and reproducible method that evaluates a physical measurement, the biochemical composition, of each unstained cell; the term " spectral cytopathology (SCP) " has been coined to describe the combination of microscopic infrared data acquisition and analysis of the spectral data via multivariate methods. 3−5 After IR acquisition, samples can then be subjected to traditional staining protocols and evaluated via conventional cytopathol-ogy means to compare results from both techniques. Since the early successes of SCP, many groups have begun investigating cultured cell lines to provide additional information regarding disease diagnosis and biological information. 6−8 Cultured cells serve several purposes ranging from distinguishing between different cell lines to their behavior in understanding disease and evaluation of drug effects and uptake. 8−10 Cultured cell lines offer a microscopic model system to explore and probe mechanisms, pathways, drug interactions, etc. Most importantly, diseased cells can be potentially biopsied from a patient's organ and propagated in cell culture conditions to be investigated thoroughly. 8,11 Often fixation procedures are applied to preserve cells for extended periods; however, the spectral effects of fixation on cultured cells are not entirely understood. 12 Previous reports claim fixation protocols introduce large spectral changes and obstruct proper analyses, speculating fixation methods as obstacles to be avoided. 13−15 This is the second paper in a series aimed at addressing the effects of fixation and storage conditions on spectral data of cellular samples. In the first paper, we described the influence of these factors to exfoliated oral (buccal) mucosa cells and demonstrated that exceedingly small variances occurred upon various fixation methods that were negligible in comparison to
... Depending on the tissue sample processing (e.g., paraffin embedding, freezing, chemical fixation, etc.) and the data analysis (e.g., spectral correction, normalization, derivation, etc.), the infrared values obtained may vary. Although Mazur and collaborators showed no significant differences in the infrared output of unfixed versus formalin-fixed cells [18,19], Hackett and collaborators demonstrated some slight differences in the infrared spectrum of the cryo-fixed tissue before and Graphs represents absorbance second derivative (d 2 A) ratios of functional groups related to lipids (A-C) and proteins (D-F) in human and murine brain samples. Each dot in the graphs correspond to the mean of approximately 100 infrared measurements from a subject. ...
... Depending on the tissue sample processing (e.g., paraffin embedding, freezing, chemical fixation, etc.) and the data analysis (e.g., spectral correction, normalization, derivation, etc.), the infrared values obtained may vary. Although Mazur and collaborators showed no significant differences in the infrared output of unfixed versus formalin-fixed cells [18,19], Hackett and collaborators demonstrated some slight differences in the infrared spectrum of the cryo-fixed tissue before and after formalin fixation [20]. However, our data in both cryo-fixed and paraformaldehyde-fixed tissue show a specific composition between brain areas already reported in rats [9,15,21] and humans [11], regardless of histological sample processing. ...
... These similarities between species in non-pathological conditions make the mouse a good experimental animal model. µFTIR comparative biology studies, but also comparative sample and data processing studies [18][19][20] are essential to assess and understand differences and similarities between species, especially for disease-related animal models. Specifically, the study of lipid-related disorders in mice could be very reliable in humans due to the high reproducibility observed in the lipid characteristics of GM and WM; however, the study of protein-related pathologies in mouse models requires a proper assessment sample and data processing variables. ...
Fourier Transform Infrared microspectroscopy (μFTIR) is a very useful method to analyze the biochemical properties of biological samples in situ. Many diseases affecting the central nervous system (CNS) have been studied using this method, to elucidate alterations in lipid oxidation or protein aggregation, among others. In this work, we describe in detail the characteristics between grey matter (GM) and white matter (WM) areas of the human brain by μFTIR, and we compare them with the mouse brain (strain C57BL/6), the most used animal model in neurological disorders. Our results show a clear different infrared profile between brain areas in the lipid region of both species. After applying a second derivative in the data, we established a 1.5 threshold value for the lipid/protein ratio to discriminate between GM and WM areas in non-pathological conditions. Furthermore, we demonstrated intrinsic differences of lipids and proteins by cerebral area. Lipids from GM present higher C=CH, C=O and CH3 functional groups compared to WM in humans and mice. Regarding proteins, GM present lower Amide II amounts and higher intramolecular β-sheet structure amounts with respect to WM in both species. However, the presence of intermolecular β-sheet structures, which is related to β-aggregation, was only observed in the GM of some human individuals. The present study defines the relevant biochemical properties of non-pathological human and mouse brains by μFTIR as a benchmark for future studies involving CNS pathological samples.
... Huang et al. 68 described the effects of formalin fixa on on RS of cancerous human bronchial ssue, whereas Draux et al. 71 described the influence of formalin and air drying on single cancer cells and a ributed spectral changes to affec on of nucleic acids and proteins. Even though not only a loss of the original chemical composi on but also poten al contamina on due to the process of formalin-fixa on in murine brain ssue was determined by Hacke et al. 76 , several studies proposed formalin fixa on as a sufficient and favorable method for subsequent spectroscopic diagnos c 77,78 . As a proof of concept, Stefanakis et al. 79 demonstrated the feasibility of vibra onal spectroscopy on formalin-fixed malignant brain ssue. ...
In recent years, Raman spectroscopy has been more and more frequently applied to address research questions in neuroscience. As a non-destructive technique based on inelastic scattering of photons, it can be used for a wide spectrum of applications including neurooncological tumor diagnostics or analysis of misfolded protein aggregates involved in neurodegenerative diseases. Progress in the technical development of this method allows for an increasingly detailed analysis of biological samples and may therefore open new fields of applications. The goal of our review is to provide an introduction into Raman scattering, its practical usage and also commonly associated pitfalls. Furthermore, intraoperative assessment of tumor recurrence using Raman based histology images as well as the search for non-invasive ways of diagnosis in neurodegenerative diseases are discussed. Some of the applications mentioned here may serve as a basis and possibly set the course for a future use of the technique in clinical practice. Covering a broad range of content, this overview can serve not only as a quick and accessible reference tool but also provide more in-depth information on a specific subtopic of interest.
... 27 Providing that one fixation method is consistently maintained throughout the study, fixationinduced spectral effects can be minimized, thereby permitting useful comparisons between different cell types and disease states. 28 For instance, Raman microscopy in conjunction with linear discriminate analyses successfully differentiated between erythrocyte, leukocyte, acute myeloid leukemia, and breast tumor cells trapped inside microfluidic devices with high accuracies. 29 Moreover, the study showed that accuracies were comparable to previous studies in which the same cell types were air-dried or fixed on Petri dishes. ...
Gene engineering is a commonly used tool in cellular biology to determine changes in function or expression of downstream targets. However, the impact of genetic modulation on biochemical effects is less frequently evaluated. The aim of this study is to use Raman microscopy to assess the biochemical effects of gene silencing on T24 and UMUC-13 bladder cancer cell lines. Cellular biochemical information related to nucleic acid and lipogenic components was obtained from deconvolved Raman spectra. We show that the green fluorescence protein (GFP), the chromophore that served as a fluorescent reporter for gene silencing, could also be detected by Raman microscopy. Only the gene-silenced UMUC-13 cell lines exhibited low-to-moderate GFP fluorescence as determined by fluorescence imaging and Raman spectroscopic studies. Moreover, we show that gene silencing and cell phenotype had a greater effect on nucleic acid and lipogenic components with minimal interference from GFP expression. Gene silencing was also found to perturb cellular protein secondary structure in which the amount of disorderd protein increased at the expense of more ordered protein. Overall, our study identified the spectral signature for cellular GFP expression and elucidated the effects of gene silencing on cancer cell biochemistry and protein secondary structure. © 2016 Society of Photo-Optical Instrumentation Engineers (SPIE).
... Previously it has been argued that fixation induced effects are inherently reproduced sample-wide and are not significant enough to confound disease-state changes or other differences being examined. 43,44 However, we have shown here that differences between experiments arise even when the same optimized fixation protocol is carried out by the same individual, on the same day, in the same lab, thus demonstrating the irreproducibility of fixation due to the sheer number of variables at play. While these differences are indeed relatively small and would often be less than those caused by disease, environment, etc., they limit the sensitivity of such experiments in the same manner as the within-sample variation of unsynchronised samples imposed by the cell cycle. ...
Single molecule localization microscopy (SMLM) and Synchrotron Fourier transform infrared (S-FTIR) spectroscopy are two techniques capable of elucidating unique and valuable biological detail. SMLM provides images of the structures and distributions of targeted biomolecules at spatial resolutions up to an order of magnitude better than the diffraction limit, whereas S-FTIR spectroscopy objectively measures the holistic biochemistry of an entire sample thereby revealing any variations in overall composition. Both tools are currently applied extensively to detect cellular response to disease, chemical treatment and environmental change. Here, these two techniques have been applied correlatively at the single cell level to probe the biochemistry of common fixation methods and have detected various fixation-induced losses of biomolecular composition and cellular ultrastructure. Furthermore, by extensive honing and optimizing of fixation protocols, many fixation artifacts previously considered pervasive and regularly identified using FTIR spectroscopy and fluorescence techniques, have been avoided. Both paraformaldehyde and two-step glutaraldehyde fixation were identified as best preserving biochemistry for both SMLM and FTIR studies while other glutaraldehyde and methanol fixation protocols were demonstrated to cause significant biochemical changes and higher variability between samples. Moreover, the potential complementarity of the two techniques was strikingly demonstrated in the correlated detection of biochemical changes as well as in the detection of fixation-induced damage that was only revealed by one of the two techniques.
... However, it has become apparent that how the tissue is handled prior to FTIR imaging requires a number of considerations that are relatively unimportant when using conventional microscopy methods [22][23][24]. These include sample thickness, hydration and the interference caused by external substances such as dyes and culture media [3]. ...
Fourier Transform Infrared (FTIR) micro-spectroscopy is an emerging technique for the biochemical analysis of tissues and cellular materials. It provides objective information on the holistic biochemistry of a cell or tissue sample and has been applied in many areas of medical research. However, it has become apparent that how the tissue is handled prior to FTIR micro-spectroscopic imaging requires special consideration, particularly with regards to methods for preservation of the samples. We have performed FTIR micro-spectroscopy on rodent heart and liver tissue sections (two spectroscopically very different biological tissues) that were prepared by desiccation drying, ethanol substitution and formalin fixation and have compared the resulting spectra with that of fully hydrated freshly excised tissues. We have systematically examined the spectra for any biochemical changes to the native state of the tissue caused by the three methods of preparation and have detected changes in infrared (IR) absorption band intensities and peak positions. In particular, the position and profile of the amide I, key in assigning protein secondary structure, changes depending on preparation method and the lipid absorptions lose intensity drastically when these tissues are hydrated with ethanol. Indeed, we demonstrate that preserving samples through desiccation drying, ethanol substitution or formalin fixation significantly alters the biochemical information detected using spectroscopic methods when compared to spectra of fresh hydrated tissue. It is therefore imperative to consider tissue preparative effects when preparing, measuring, and analyzing samples using FTIR spectroscopy.
Key events in postnatal brain development, such as neuronal migration, synaptogenesis, and myelination, shape the adult brain. These events are reflected in changes in gray and white matter (GM and WM) occurring during this period. Therefore, precise knowledge of GM and WM composition in perinatal brain development is crucial to characterizing brain formation as well as the neurodevelopmental disruption observed in diseases such as autism and schizophrenia. In this study, we combined histochemical and immunohistochemical staining with biochemical and biophysical analyses using Fourier transform infrared (IR) microspectroscopy (μFTIR) to better understand the chemical changes during postnatal developmental myelination. For this purpose, we analyzed the GM and WM in the mouse brain and cerebellum (strain C57BL/6) from postnatal day 0 (P0) to day P28 and established presumed correlations between staining and IR data. IR spectra allowed the (i) quantification of lipid and protein content through the CH2/amide I ratio, (ii) determination of chemical characteristics of lipids, such as the presence of unsaturated bonds in the carbonate chain or carbonyls from ester groups in the polar head, and (iii) determination of the protein secondary structure (α-helix and intramolecular β-sheets). The results indicate that the increase in the CH2/amide I ratio calculated from the μFTIR data correlates well with lipid histochemical staining. IR data indicated a change in the lipid composition in WM since carbonyl and unsaturated olefinic groups do not increase when lipids accumulate during myelination. Our correlation analysis between IR data and immunohistochemical staining of myelin-associated proteins revealed that myelin oligodendrocyte protein correlated well with lipid accumulation, while myelin basic protein appeared before lipid modifications, which indicated that myelin-associated proteins and lipid deposition were not synchronic. These events were related to a decrease in the intramolecular β/α protein ratio. Our results indicate that lipids and proteins in WM substantially change their composition due to primary myelination, and according to results obtained from staining, these modifications are better described by lipid histochemical staining than by immunohistochemistry against myelin-related proteins. In conclusion, μFTIR can be a useful technique to study WM during perinatal development and provide detailed information about alterations in the chemical composition related to neurodevelopmental diseases.
Herein, a technique to analyze air-dried kidney tissue impression smears by means of Attenuated Total Reflection Infrared (ATR-IR) spectroscopy is presented. Spectral tumor markers - absorption bands of glycogen - are identified in the ATR-IR spectra of the kidney tissue smear samples. Thin kidney tissue cryo-sections currently used for IR spectroscopic analysis lack such spectral markers as the sample preparation causes irreversible molecular changes in the tissue. In particular, freeze-thaw cycle results in degradation of the glycogen and reduction or complete dissolution of its content. Supervised spectral classification was applied to the recorded spectra of the smears and the test spectra were classified with a high accuracy of 92 % for normal tissue and 94 % for tumor tissue, respectively. For further development, we propose that combination of the method with optical fiber ATR probes could potentially be used for rapid real-time intra-operative tissue analysis without interfering with either the established protocols of pathological examination or the ordinary workflow of operating surgeon. Such approach could ensure easier transition of the method to clinical applications where it may complement the results of gold standard histopathology examination and aid in more precise resection of kidney tumors.
As asserted by numerous authors, Fourier Transform (FT)-Raman and FT-Infrared spectroscopies
constitute two useful tools for bio-medical diagnostics. We can distinguish two
different kinds of application, namely, tissue disorders analyzed using statistical methods,
and detection of ectopic calcifications through precise analysis of the shape and the position
of absorption bands. In this contribution, we present some new results regarding
these research themes, as well as recent experimental developments, such as the ability to
perform nanometer scale near-field infrared microscopy, which have already led to major
scientific breakthroughs.
During the last 15years, vibrational spectroscopic methods have been developed, which can be viewed as molecular pathology methods that depend on sampling the entire genome, proteome, and metabolome of cells and tissues, rather than probing for the presence of selected markers. First, this review introduces the background and fundamentals of the spectroscopies underlying the new methodologies, namely infrared and Raman spectroscopy. Then, results are presented in the context of spectral histopathology of tissues for the detection of metastases in lymph nodes, squamous cell carcinoma, adenocarcinomas, brain tumors, and brain metastases. Results from spectral cytopathology of cells are discussed for screening of oral and cervical mucosa and circulating tumor cells. It is concluded that infrared and Raman spectroscopy can complement histopathology and reveal information that is available in classical methods only by costly and time-consuming steps such as immunohistochemistry, polymerase chain reaction, or gene arrays. Because of the inherent sensitivity toward changes in the biomolecular composition of different cell and tissue types, vibrational spectroscopy can even provide information that is in some cases superior to that obtained by any one of the conventional techniques.
Infrared imaging was applied to investigate a reconstituted basement membrane, known as Matrigel, in three-dimensional cell cultures. Matrigel, in the vicinity of the colonies, was examined for four breast cancer cell lines presenting different 3D colony morphologies. MCF-7 and T-47D present mass colonies, SKBR-3 grape-like colonies and MDA-MB-231 stellate colonies associated with a more invasive phenotype. The edge of the cell colonies was found to be significantly depleted in Matrigel. Except in a limited number of cases, Matrigel appeared to be thinner at the edges of the colonies but not completely destroyed or torn off as it would be for a purely mechanical effect. When a PCA was run on the spectra of one or several colonies, the score images on PC#3 and PC#4 presented structures in the Matrigel areas which appeared as fringes, lines, dots or regular patterns. This effect represents a very small fraction of the total variance but is reproducible for all the 4 cell lines. PC#4 presents systematically a maximum near 1624 cm(-1) and a minimum around 1700 cm(-1). When spectra are normalized, the effect is less marked but does not disappear. The nature of the variations that exist in the Matrigel layer is therefore not solely related to thickness but also to the chemical composition. At this stage, the weakness of the effect prevents a thorough investigation.
Alkaloid applications can be found in different areas and across history. Some alkaloids are still used in chemistry and modern medicine as natural or modified compounds. Their use is connected to the regulation of Na⁺ ions and channels, mescaric, cholinergic receptors, acetylcholine esterase, opiod and opiate receptors, glycine, 5-HTRs, and other receptors, as well as the regulation of microtubules of the spindle apparatus, antiproliferative activity, inhibition of some enzymes, and induction of apoptosis. Moreover, alkaloids are used in the regulation of microbial and schizonticide activity and as pharmaceuticals. Alkaloids are also generally a problem in food; there are some applications in agriculture, especially in plant breeding (alkaloid-rich and alkaloid-poor cultivars). Genetically modified organisms (GMOs) can be considered to hold possibilities for vaccine development, especially in plants. Some alkaloids are used in food receptors as additional components or are consumed as a part of the final product (caffeine, theophylline, piperine, capsaicin). The use of alkaloids as a supplement in some products on the market is presently a matter of discussion, as they are considered a health risk (the case of ephedrine). Alkaloids can be used as biological fertilizers and control agents in plant protection. Biotechnology opens new possibilities of alkaloid applications. The productional achievements are found in cell and organ cultures. The most well-known applications in root cultures are with anabasine, nicotine, harmine, harmaline, hyoscyamine, calystegine, scopolamine, and senecionine. Alkaloid enzymes can be purified from these cultures. Bioreactors and blue, white, and fungal biotechnologies are nowadays used in industrial production.
This review will take a fresh approach from the patient perspective; offering insight into the applications of mid-infrared biomedical spectroscopy in a scenario whereby the patient presents with non-specific symptoms and via an extensive diagnostic process multiple lesions are discovered but no clear sign of the primary tumour; a condition known as cancer of unknown primary (CUP). With very limited options to diagnose the cancer origin, treatment options are likely to be ineffective and prognosis is consequentially very poor. CUP has not yet been targeted by infrared biospectroscopy, however, this timely, concise dissemination will focus on a series of research highlights and breakthroughs from the field for the management of a variety of cancer-related diseases - many examples of which have occurred within this year alone. The case for integration of mid-infrared (MIR) technology into clinical practice will be demonstrated largely via diagnostic, but also therapeutic and prognostic avenues by means of including cytological, bio-fluid and tissue analysis. The review is structured around CUP but is relevant for all cancer diagnoses. Infrared spectroscopy is fast developing a reputation as a valid and powerful tool for the detection and diagnosis of cancer using a variety of sample formats. The technology will produce data and tools that are designed to complement routine clinical practice; enhancing the ability of the clinician to make a reliable and non-subjective decision and enabling decreased levels of mortality and morbidity and gains in patient quality of life.
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.
Instrumental advances in vibrational microspectroscopy 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 chapter is to summarize the progress achieved since the last edition of this book in using spectral cytopathology (SCP) – the combination of infrared (IR) microspectroscopy and multivariate methods of analysis – for the detection of abnormalities in exfoliated human cells. This work sets the stage for biomedical and diagnostic applications of this technology.
Results of a study comparing infrared imaging data sets collected on different instruments or instrument platforms are reported, along with detailed methods developed to permit such comparisons. It was found that different instrument platforms, although employing different detector technologies and pixel sizes, produce highly similar and reproducible spectral results. However, differences in the absolute intensity values of the reflectance data sets were observed that were cause by heterogeneity of the sample sub-strate in terms of reflectivity and planarity.
Fixation of biological sample is an essential technique applied in order to "freeze" in time the intracellular molecular content and permit mapping of cellular molecules. However, fixation induces changes of the cellular molecular structure, which mask physiological distribution of biomolecules and bias interpretation of results. Accurate, sensitive, and comprehensive characterization of changes in biomolecular composition, occurring during fixation, is crucial for proper analysis of experimental data. Here we apply Biomolecular Component Analysis for Raman spectra measured in the same nucleoli of HeLa cells before and after fixation by either formaldehyde solution or by chilled ethanol. It is found that fixation in formaldehyde does not strongly effect the Raman spectra of nucleolar biomolecular components, but may significantly decrease the nucleolar RNA concentration. At the same time, ethanol fixation leads to a proportional increase (up to 40%) in concentrations of nucleolar proteins and RNA, most likely due to cell shrinkage occurring in the presence of coagulant fixative. Ethanol fixation also triggers changes in composition of nucleolar proteome, as indicated by an overall reduction of the α-helical structure of proteins and increase in the concentration of proteins containing the β-sheet conformation. We conclude that cross-linking fixation is a more appropriate protocol for mapping of proteins in situ. At the same time, ethanol fixation is preferential for studies of RNA-containing macromolecules. We supplemented our quantitative Raman spectroscopic measurements with mapping of the protein and lipid macromolecular groups in live and fixed cells using Coherent Anti-Stokes Raman Scattering nonlinear optical imaging.
During the last 15 years, vibrational spectroscopic methods have been developed that can be viewed as molecular pathology methods that depend on sampling the entire genome, proteome and metabolome of cells and tissues, rather than probing for the presence of selected markers. First, this review introduces the background and fundamentals of the spectroscopies underlying the new methodologies, namely infrared and Raman spectroscopy. Then, results are presented in the context of spectral histopathology of tissues for detection of metastases in lymph nodes, squamous cell carcinoma, adenocarcinomas, brain tumors and brain metastases. Results from spectral cytopathology of cells are discussed for screening of oral and cervical mucosa, and circulating tumor cells. It is concluded that infrared and Raman spectroscopy can complement histopathology and reveal information that is available in classical methods only by costly and time-consuming steps such as immunohistochemistry, polymerase chain reaction or gene arrays. Due to the inherent sensitivity toward changes in the bio-molecular composition of different cell and tissue types, vibrational spectroscopy can even provide information that is in some cases superior to that of any one of the conventional techniques. (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).
Circulating tumor cells present an important marker of the progress of several cancer diseases including breast and colorectal cancer, and enables an interesting prognosis and diagnostic options that can complement convenient diagnostic techniques based on several imaging methods. Based on its relevance, the analysis of such kinds of cells is within the scope of many research and clinical institutes; however, it still presents a difficult task. Here we used a state-of-the-art micro-Raman microscopic technique to enhance possibilities in the study of circulating tumor cells and their further differentiation. As cytospins present a convenient form of sample collection and preparation, we used this form of sampling as the initial point. Raman presents a non-destructive form of sample analysis and thus the samples can be further used for a method validation. We have considered the influence of fixation methods of the cells, where we found out that the ability of Raman spectroscopy to differentiate between three cell lines strictly depends upon the sample preparation method used. Namely breast (BT 549) and colorectal (HCT 116) circulating tumor cell lines and human mononuclear cells were compared. Methanol and paraformaldehyde methods of fixation were compared to simple drying out of the sample. It has been shown that drying out of the cells enables the best performance to be obtained in cell differentiation and this is demonstrated by the use of principal component analysis, where all three given cell lines were differentiated with a high level of confidence. Next, the cells were also scanned using 1 μm spatial resolution. The acquired data were visualized using both chemigrams and hierarchical clustering analysis.
Over the past few decades, Fourier transform infrared (FTIR) spectroscopy coupled to microscopy has been recognized as an emerging and potentially powerful tool in cancer research and diagnosis. For this purpose, histological analyses performed by pathologists are mostly carried out on biopsied tissue that undergoes the formalin-fixation and paraffin-embedding (FFPE) procedure. This processing method ensures an optimal and permanent preservation of the samples, making FFPE-archived tissue an extremely valuable source for retrospective studies. Nevertheless, as highlighted by previous studies, this fixation procedure significantly changes the principal constituents of cells, resulting in important effects on their infrared (IR) spectrum. Despite the chemical and spectral influence of FFPE processing, some studies demonstrate that FTIR imaging allows precise identification of the different cell types present in biopsied tissue, indicating that the FFPE process preserves spectral differences between distinct cell types. In this study, we investigated whether this is also the case for closely related cell lines. We analyzed spectra from 8 cancerous epithelial cell lines: 4 breast cancer cell lines and 4 melanoma cell lines. For each cell line, we harvested cells at subconfluence and divided them into two sets. We first tested the "original" capability of FTIR imaging to identify these closely related cell lines on cells just dried on BaF2 slides. We then repeated the test after submitting the cells to the FFPE procedure. Our results show that the IR spectra of FFPE processed cancerous cell lines undergo small but significant changes due to the treatment. The spectral modifications were interpreted as a potential decrease in the phospholipid content and protein denaturation, in line with the scientific literature on the topic. Nevertheless, unsupervised analyses showed that spectral proximities and distances between closely related cell lines were mostly, but not entirely, conserved after FFPE processing. Finally, PLS-DA statistical analyses highlighted that closely related cell lines are still successfully identified and efficiently distinguished by FTIR spectroscopy after FFPE treatment. This last result paves the way towards identification and characterization of cellular subtypes on FFPE tissue sections by FTIR imaging, indicating that this analysis technique could become a potential useful tool in cancer research.
This review deals with the role of carboxylic amino acids in the proton-transport activity of bacteriorhodopsin. The main focus is on the infrared data, which allow direct monitoring of the protonation/deprotonation of specific residues during the proton movement in the course of the photocycle. Additional attention is paid to the potential use of carboxylic acids in proteins as internal sensors, based on the sensitivity of their IR frequencies to the immediate environment.
Aim
Spectral Cytopathology (SCP) is a novel spectroscopic method for objective and unsupervised classification of individual exfoliated cells. The limitations of conventional cytopathology are well-recognized within the pathology community. In SCP, cellular differentiation is made by observing molecular changes in the nucleus and the cytoplasm, which may or may not produce morphological changes detectable by conventional cytopathology. This proof of concept study demonstrates SCP’s potential as an enhancing tool for cytopathologists by aiding in the accurate and reproducible diagnosis of cells in all states of disease.
Method
Infrared spectra are collected from cervical cells deposited onto reflectively coated glass slides. Each cell has a corresponding infrared spectrum that describes its unique biochemical composition. Spectral data are processed and analyzed by an unsupervised chemometric algorithm, Principal Component Analysis (PCA).
Results
In this blind study, cervical samples are classified by analyzing the spectra of morphologically normal looking squamous cells from normal samples and samples diagnosed by conventional cytopathology with low grade squamous intraepithelial lesions (LSIL). SCP discriminated cytopathological diagnoses amongst twelve different cervical samples with a high degree of specificity and sensitivity. SCP also correlated two samples with abnormal spectral changes: these samples had a normal cytopathological diagnosis but had a history of abnormal cervical cytology. The spectral changes observed in the morphologically normal looking cells are most likely due to an infection with human papillomavirus, HPV. HPV DNA testing was conducted on five additional samples, and SCP accurately differentiated these samples by their HPV status.
Conclusions
SCP tracks biochemical variations in cells that are consistent with the onset of disease. HPV has been implicated as the cause of these changes detected spectroscopically. SCP does not depend on identifying the sparse number of morphologically abnormal cells within a large sample in order to make an accurate classification, as does conventional cytopathology. These findings suggest that the detection of cellular biochemical variations by SCP can serve as a new enhancing screening method that can identify earlier stages of disease.
Spectral cytopathology (SCP) is a novel approach for diagnostic differentiation of disease in individual exfoliated cells. SCP is carried out by collecting information on each cell's biochemical composition through an infrared micro-spectral measurement, followed by multivariate data analysis. Deviations from a cell's natural composition produce specific spectral patterns that are exclusive to the cause of the deviation or disease. These unique spectral patterns are reproducible and can be identified and used through multivariate statistical methods to detect cells compromised at the molecular level by dysplasia, neoplasia, or viral infection. In this proof of concept study, a benchmark for the sensitivity of SCP is established by classifying healthy oral squamous cells according to their anatomical origin in the oral cavity. Classification is achieved by spectrally detecting cells with unique protein expressions: for example, the squamous cells of the tongue are the only cell type in the oral cavity that have significant amounts of intracytoplasmic keratin, which allows them to be spectrally differentiated from other oral mucosa cells. Furthermore, thousands of cells from a number of clinical specimens were examined, among them were squamous cell carcinoma, malignancy-associated changes including reactive atypia, and infection by the herpes simplex virus. Owing to its sensitivity to molecular changes, SCP often can detect the onset of disease earlier than is currently possible by cytopathology visualization. As SCP is based on automated instrumentation and unsupervised software, it constitutes a diagnostic workup of medical samples devoid of bias and inconsistency. Therefore, SCP shows potential as a complementary tool in medical cytopathology.
Synchrotron-based Fourier transform infrared (SR-FTIR) microspectroscopy is a powerful bioanalytical technique for the simultaneous analysis of lipids, proteins, carbohydrates, and a variety of phosphorylated molecules within intact cells. SR-FTIR microspectroscopy can be used in the imaging mode to generate biospectroscopic maps of the distribution and intensity profiles of subcellular biomolecular domains at diffraction-limited spatial resolution. However, the acquisition of highly spatially resolved IR images of cells is not only a function of instrumental parameters (source brightness, sampling aperture size) but also the cell preparation method employed. Additionally, for the IR data to be biochemically relevant the cells must be preserved in a life-like state without introducing artefacts. In the present study we demonstrate, for the first time, the differences in biomolecular localizations observed in SR-FTIR images of cells fixed by formalin, formalin-critical point drying (CPD), and glutaraldehyde-osmium tetroxide-CPD, using the PC-3 prostate cancer cell line. We compare these SR-FTIR images of fixed cells to unfixed cells. The influence of chemical fixatives on the IR spectrum is discussed in addition to the biological significance of the observed localizations. Our experiments reveal that formalin fixation at low concentration preserves lipid, phosphate, and protein components without significantly influencing the IR spectrum of the cell.
A transformation known as the maximum noise fraction (MNF)
transformation, which always produces new components ordered by image
quality, is presented. It can be shown that this transformation is
equivalent to principal components transformations when the noise
variance is the same in all bands and that it reduces to a multiple
linear regression when noise is in one band only. Noise can be
effectively removed from multispectral data by transforming to the MNF
space, smoothing or rejecting the most noisy components, and then
retransforming to the original space. In this way, more intense
smoothing can be applied to the MNF components with high noise and low
signal content than could be applied to each band of the original data.
The MNF transformation requires knowledge of both the signal and noise
covariance matrices. Except when the noise is in one band only, the
noise covariance matrix needs to be estimated. One procedure for doing
this is discussed and examples of cleaned images are presented
Chemometrics in Analytical Spectroscopy provides students and practising analysts with a tutorial guide to the use and application of the more commonly encountered techniques used in processing and interpreting analytical spectroscopic data. In detail the book covers the basic elements of univariate and multivariate data analysis, the acquisition of digital data and signal enhancement by filtering and smoothing, feature selection and extraction, pattern recognition, exploratory data analysis by clustering, and common algorithms in use for multivariate calibration techniques. An appendix is included which serves as an introduction or refresher in matrix algebra. The extensive use of worked examples throughout gives Chemometrics in Analytical Spectroscopy special relevance in teaching and introducing chemometrics to undergraduates and post-graduates undertaking analytical science courses. It assumes only a very moderate level of mathematics, making the material far more accessible than other publications on chemometrics. The book is also ideal for analysts with little specialist background in statistics or mathematical methods, who wish to appreciate the wealth of material published in chemometrics.
In attempting to analyze, on digital computers, data from basically continuous physical experiments, numerical methods of performing familiar operations must be developed. The operations of differentiation and filtering are especially important both as an end in themselves, and as a prelude to further treatment of the data. Numerical counterparts of analog devices that perform these operations, such as RC filters, are often considered. However, the method of least squares may be used without additional computational complexity and with considerable improvement in the information obtained. The least squares calculations may be carried out in the computer by convolution of the data points with properly chosen sets of integers. These sets of integers and their normalizing factors are described and their use is illustrated in spectroscopic applications. The computer programs required are relatively simple. Two examples are presented as subroutines in the FORTRAN language.
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 objective of the present study was to establish the conditions for infrared spectroscopy of single living cells in cell culture under aqueous media, using a high brilliance synchrotron light source to achieve acceptable S/N at the necessary high spatial resolution. This is experimentally challenging, but brings a number of significant advantages. The data presented show that the infrared spectra of single living cells can be monitored over several hours with high reproducibility, as well as confirming that acquisition of spectral data from single cells can contribute new insights that are not available from measurements of cell populations.
Spectral differences between normal and abnormal tissue observed to date appear to be due to different averaging processes of spectral patterns that differ according to the cell's biochemistry, due to its state of maturation, differentiation, and development. Thus, disease perturbs the distribution of cells in the different stages of maturation, differentiation, and development. Previous FTIR microspectroscopic studies of normal versus neoplastic cells and tissues have demonstrated differences in the absorption intensities and band-shapes, particularly in the low frequency (1200–1000 cm−1) spectral region. In this study, we further investigated the spectral changes due to the drastic biochemical and morphological changes occurring as a consequence of cell proliferation.
In this paper we describe the advantages of collecting infrared microspectral data in imaging mode opposed to point mode. Imaging data are processed using the PapMap algorithm, which co-adds pixel spectra that have been scrutinized for R-Mie scattering effects as well as other constraints. The signal-to-noise quality of PapMap spectra will be compared to point spectra for oral mucosa cells deposited onto low-e slides. Also the effects of software atmospheric correction will be discussed. Combined with the PapMap algorithm, data collection in imaging mode proves to be a superior method for spectral cytopathology. (© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)
After two decades of intense research on the spectroscopic properties of biological molecules in isolated systems, infrared spectroscopy is now being applied to the study of human tissues. Extending this approach, it is possible to use the sensitivity of infrared spectroscopy to probe the biochemical events underlying transformation from normal to a diseased state within tissues, and so develop novel diagnostic methods. We highlight some of the areas of research within our group aimed at developing clinically useful methodologies based upon infrared spectroscopy.
We have optimized an imaging methodology capable of monitoring individual live HeLa cells using non-synchrotron FTIR in an aqueous environment. This methodology, in combination with MATLAB based pre-processing techniques, allows fast and efficient collection of data with high signal-to-noise ratio in comparison with previous methods using point mode data collection, which required manual operation and more collection time. Also, presented are early results that illustrate interpretable spectral differences from live cells treated with chemotherapeutic drugs, demonstrating the potential of this methodology to develop more desirable modes of treatment for patients in their diagnoses and treatments for disease.
Spectral cytopathology (SCP) is a novel approach for disease diagnosis that utilizes infrared spectroscopy to interrogate the biochemical components of cellular samples and multivariate statistical methods, such as principal component analysis, to analyze and diagnose spectra. SCP has taken vast strides in its application for disease diagnosis over the past decade; however, fixation-induced changes and sample handling methods are still not systematically understood. Conversely, fixation and staining methods in conventional cytopathology, typically involving protocols to maintain the morphology of cells, have been documented and widely accepted for nearly a century. For SCP, fixation procedures must preserve the biochemical composition of samples so that spectral changes significant to disease diagnosis are not masked. We report efforts to study the effects of fixation protocols commonly used in traditional cytopathology and SCP, including fixed and unfixed methods applied to exfoliated oral (buccal) mucosa cells. Data suggest that the length of time in fixative and duration of sample storage via desiccation contribute to minor spectral changes where spectra are nearly superimposable. These findings illustrate that changes influenced by fixation are negligible in comparison to changes induced by disease.
Fourier Transform Infrared (FTIR) spectroscopic measurements of individual, live HeLa cells in culture and buffer media are presented. Spectral data were acquired using a newly designed live cell chamber developed in the authors' laboratory. Data were processed using MATLAB-based routines that correct for the overcompensation of water encountered during live cell measurements in aqueous samples. Data presented are from live cells monitored over an extended period of time as well as a comparison of live cells exposed to perturbing conditions.
Fourier Transform Infrared (FT-IR) spectroscopic imaging is emerging as an automated alternative to human examination in studying development and disease in tissue. The technology's speed and accuracy, however, are limited by the trade-off with signal-to-noise ratio (SNR). Signal processing approaches to reduce noise have been suggested but often involve manual decisions, compromising the automation benefits of using spectroscopic imaging for tissue analysis. In this manuscript, we describe an approach that utilizes the spatial information in the data set to select parameters for noise reduction without human input. Specifically, we expand on the Minimum Noise Fraction (MNF) approach in which data are forward transformed, eigenimages that correspond mostly to signal selected and used in inverse transformation. Our unsupervised eigenimage selection method consists of matching spatial features in eigenimages with a low-noise gold standard derived from the data. An order of magnitude reduction in noise is demonstrated using this approach. We apply the approach to automating breast tissue histology, in which accuracy in classification of tissue into different cell types is shown to strongly depend on the SNR of data. A high classification accuracy was recovered with acquired data that was ∼10-fold lower SNR. The results imply that a reduction of almost two orders of magnitude in acquisition time is routinely possible for automated tissue classifications by using post-acquisition noise reduction.
Raman spectroscopy provides chemical-rich information about the composition of analytes and is a powerful tool for biological studies. With the ability to investigate specific cellular components or image whole cells, compatible methods of sample preservation must be implemented for accurate spectra to be collected. Unfortunately, the effects of many commonly used sample preservation methods have not been explored with cultured cells. In this study, two human cell lineages of varying phenotypes were used to investigate the effects of sample preservation methods. Cells were cultured directly onto quartz substrates and either formalin-fixed, desiccated or air dried. The results indicate that the methodology applied to cell cultures for Raman analysis significantly influences the quality and reproducibility of the resulting spectral data. Formalin fixation was not found to be as universally efficient as anticipated for a commonly used fixative. This was due largely to the inconsistency in sample preservation between cell lines and loss of signal intensity. Sample air-drying was found to be largely inconsistent in terms of spectral reproducibility. Our study shows that sample desiccation displayed good spectral reproducibility and resulted in a good signal-to-noise ratio. Lipid and protein content in both activated and inactivated cells were maintained and provided a more controlled method compared with air-drying, revealing that the speed of drying is important for sample preservation.
Infrared absorption spectroscopy has been used to study the effect of organic solvents on the conformation of myoglobin, apomyoglobin, hemoglobin, lysozyme and ribonuclease. Beta structure can easily be induced by specific solvent effects. Films prepared from a 50% (v/v) mixture of alcohol, acetone, pyridine, tetrahydrofuran or dimethylsulfoxide/water mixtures show a high proportion of beta structure. The degree of induction of beta structure depends on the hydrocarbon content of the alcohol in the order methanol greater than ethanol greater than butanol. No beta structure was observed in films prepared from aqueous octanol solutions. Lyophilization tends to decrease secondary structure. The conformation of the proteins depends on the particular solvent system and the solvent composition. Solution studies of myoglobin in pure dimethylsulfoxide show that the conformation is a mixture of random and beta forms while in dimethylsulfoxide/2H2O mixtures the conformation is a mixture of alpha-helical and beta forms.
The combination of synchrotron IR microspectroscopy and fluorescence microscopy has led to the identification of specific IR signatures of apoptosis and necrosis at a single cell level. Apoptosis was induced by treatment of Fas+ tumor cell lines with anti-Fas monoclonal antibodies. Detection of the early and late stages of apoptosis was performed using conjugated annexin V-fluorescein isothiocyanate (AV-FITC) and propidium iodide. Very early cellular changes were detected by IR before externalization of phosphatidylserine and AV-FITC labeling, and they were probably linked to DNA unwinding. The IR signals at 1044, 1177, and 1222 cm(-1), as well as an intensity variation in the CHx stretching region, are the main signature changes of early and late apoptosis, in line with the hypothesis of DNA fragmentation. The increased intensity of the CHx stretching bands of the lipids was observed only at an early stage of apoptosis. Changes in the relative intensity of CH3 and CH2 stretching accompany this increased intensity, suggesting changes in the relative amount and/or type of lipids concomitant with an increased lipid content. Finally, necrotic cells were characterized by marked changes in their chemical composition because several new vibrational features were observed.
We have previously reported spectral differences for cells at different stages of the eukaryotic cell division cycle. These differences are due to the drastic biochemical and morphological changes that occur as a consequence of cell proliferation. We correlate these changes in FTIR absorption and Raman spectra of individual cells with their biochemical age (or phase in the cell cycle), determined by immunohistochemical staining to detect the appearance (and subsequent disappearance) of cell-cycle-specific cyclins, and/or the occurrence of DNA synthesis. Once spectra were correlated with their cells' staining patterns, we used methods of multivariate statistics to analyze the changes in cellular spectra as a function of cell cycle phase.
We report results from a study of human and canine mucosal cells, investigated by infrared micro-spectroscopy, and analyzed by methods of multivariate statistics. We demonstrate that the infrared spectra of individual cells are sensitive to the stage of maturation, and that a distinction between healthy and diseased cells will be possible. Since this report is written for an audience not familiar with infrared micro-spectroscopy, a short introduction into this field is presented along with a summary of principal component analysis.
Fourier transform infrared (FT-IR) spectroscopy is a valuable technique for characterization of biological samples, providing a detailed fingerprint of the major chemical constituents. However, water vapor and CO(2) in the beam path often cause interferences in the spectra, which can hamper the data analysis and interpretation of results. In this paper we present a new method for removal of the spectral contributions due to atmospheric water and CO(2) from attenuated total reflection (ATR)-FT-IR spectra. In the IR spectrum, four separate wavenumber regions were defined, each containing an absorption band from either water vapor or CO(2). From two calibration data sets, gas model spectra were estimated in each of the four spectral regions, and these model spectra were applied for correction of gas absorptions in two independent test sets (spectra of aqueous solutions and a yeast biofilm (C. albicans) growing on an ATR crystal, respectively). The amounts of the atmospheric gases as expressed by the model spectra were estimated by regression, using second-derivative transformed spectra, and the estimated gas spectra could subsequently be subtracted from the sample spectra. For spectra of the growing yeast biofilm, the gas correction revealed otherwise hidden variations of relevance for modeling the growth dynamics. As the presented method improved the interpretation of the principle component analysis (PCA) models, it has proven to be a valuable tool for filtering atmospheric variation in ATR-FT-IR spectra.
Optical histopathology is fast emerging as a potential tool in cancer diagnosis. Fresh tissues in saline are ideal samples for optical histopathology. However, evaluation of suitability of ex vivo handled tissues is necessitated because of severe constraints in sample procurement, handling, and other associated problems with fresh tissues. Among these methods, formalin-fixed samples are shown to be suitable for optical histopathology. However, it is necessary to further evaluate this method from the point of view discriminating tissues with minute biochemical variations. A pilot Raman and Fourier transform infrared (FTIR) microspectroscopic studies of formalin-fixed tissues normal, malignant, and after-2-fractions of radiotherapy from the same malignant cervix subjects were carried out, with an aim to explore the feasibility of discriminating these tissues, especially the tissues after-2-fractions of radiotherapy from other two groups. Raman and FTIR spectra exhibit large differences for normal and malignant tissues and subtle differences are seen between malignant and after-2-fractions of radiotherapy tissues. Spectral data were analyzed by principal component analysis (PCA) and it provided good discrimination of normal and malignant tissues. PCA of data of three tissues, normal, malignant, and 2-fractions after radiotherapy, gave two clusters corresponding to normal and malignant + after-2-fractions of radiotherapy tissues. A second step of PCA was required to achieve discrimination between malignant and after-2-fractions of radiotherapy tissues. Hence, this study not only further supports the use of formalin-flxed tissues in optical histopathology, especially from Raman spectroscopy point of view, it also indicates feasibility of discriminating tissues with minute biochemical differences such as malignant and after-2-fractions of radiotherapy.
This review discusses the application of infrared spectroscopy to the study of proteins. The focus is on the mid-infrared spectral region and the study of protein reactions by reaction-induced infrared difference spectroscopy.
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