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Medical Applications of Infrared Spectral Imaging of Individual Cells
Abstract
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.
Cartilage is a connective tissue composed of mainly water, collagen (COL) and proteoglycans (PGs) including chondroitin sulfate (CS). Near-infrared (NIR) spectroscopy is adequate for examination of soft and hard tissues with large amount of water non-destructively and non-invasively. We measured tablets containing CS and COL using NIR spectroscopy to develop an evaluation method for PGs in cartilage non-destructively and non-invasively. Calibration curves were constructed using the NIR spectra of the tablets that show the quantitative linear relationship between the concentration and height of the second derivative at 4260 cm−1 for COL and at 5800 cm−1 for COL and CS. An equation to calculate the CS-to-COL ratio was derived from the calibrated slopes at 5800 and 4260 cm−1, and the utility of the equation was demonstrated by the evaluation of tablets. Moreover, we conducted an evaluation of the CS-to-COL ratio in the aqueous nucleus pulposus and annulus fibrosus, and the results were consistent with the glycosaminoglycans (GAGs)-to-COL ratios obtained through Raman spectroscopy of the same specimens. Thus, this method is adequate for evaluating PGs with large amount of water non-destructively, non-invasively and with less damage.
Understanding the doping and ion distributions in light-emitting electrochemical cells (LECs)
is required to approach a realistic conduction model, which can precisely explain the
electrochemical reactions, p-n junction formation, and ion dynamics in the active layer, and to
provide relevant information about LECs for systematic improvement of function and
manufacture. Here, Fourier-transform infrared (FTIR) microscopy is used to monitor anion
density profile and polymer structure in situ, and for time-resolved mapping of electrochemical
doping in an LEC under bias. The results are in very good agreement with the electrochemical
doping (ECD) model, with respect to ion redistribution and formation of a dynamic p-n junction
in the active layer. We also physically slow down ions by decreasing the working temperature
and study frozen-junction formation and immobilization of ions in a fixed-junction LEC device
by FTIR imaging. The obtained results show irreversibility of the ion redistribution and polymer
doping in a fixed-junction device. In addition, we demonstrate that infrared microscopy is a
useful tool for in situ characterization of electroactive organic materials.
This paper summarizes the progress achieved over the past fifteen years in applying vibrational (Raman and IR) spectroscopy to problems of medical diagnostics and cellular biology. During this time, a number of research groups have verified the enormous information content of vibrational spectra; in fact, genomic, proteomic, and metabolomic information can be deduced by decoding the observed vibrational spectra. This decoding process is aided enormously by the availability of high-power computer workstations and advanced algorithms for data analysis. Furthermore, commercial instrumentation for the fast collection of both Raman and infrared microspectral data has rendered practical the collection of images based solely on spectral data. The progress in the field has been manifested by a steady increase in the number and quality of publications submitted by established and new research groups in vibrational biological and biomedical arenas.
Mid-infrared spectra of biological matter such as tissues or microbial and eukaryotic cells measured in a transmission-type optical setup frequently show strongly distorted line shapes which arise from mixing of absorption and scattering contributions. Scattering-associated distorted line shapes may considerably complicate the analysis and interpretation of the infrared spectra and large efforts have been made to understand the mechanisms of scattering in biological matter and to compensate for spectral alterations caused by scattering. The goals of the present study were two-fold: firstly, to get a deeper understanding of the physics of scattering of biological systems and to explore how physical parameters of the scatterers such as shape, size and refractive index influence the line shape distortions observed. In this context, simulations based on the full Mie scattering formalism for spherical particles were found to be useful in explaining the characteristics of the Mie scatter-associated distortions and yielded a size criterion for the scattering particles similar to the well-known near field criterion. The second objective of the study was to investigate whether alternative optical setups allow minimisation of the effects of scattering. For this purpose, an optical system is proposed which is composed of an integrating sphere unit originally designed for diffuse reflection measurements, an off-axis DLaTGS detector to collect scattered and transmitted light components and a commercial Fourier transform infrared (FTIR) spectrometer. In the context of this study transmission type (tt-) FTIR spectra and spectra acquired by means of the integrating sphere setup (is-FTIR) were acquired from monodisperse poly(methyl) methacrylate (PMMA) microspheres of systematically varying sizes. The tt-FTIR spectral data of different PMMA particles confirmed earlier observations such as the presence of size-dependent oscillating spectral baselines, peak shifts, or derivative-like spectral line shapes. Such effects could be dramatically minimised when is-FTIR spectra were acquired by the integrating sphere unit. Utilisation of an integrating sphere is suggested as a convenient and easy-to use alternative to computer-based methods of scatter correction.
Transflection-mode FTIR spectroscopy has become a popular method of measuring spectra from biomedical and other samples due to the relative low cost of substrates compared to transmission windows, and a higher absorbance due to a double pass through the same sample approximately doubling the effective path length. In this publication we state an optical description of samples on multilayer low-e reflective substrates. Using this model we are able to explain in detail the so-called electric-field standing wave effect and rationalise the non-linear change in absorbance with sample thickness. The ramifications of this non-linear change, for imaging and classification systems, where a model is built from tissue sectioned at a particular thickness and compared with tissue of a different thickness are discussed. We show that spectra can be distorted such that classification fails leading to inaccurate tissue segmentation which may have subsequent implications for disease diagnostics applications.
Alkylating agents have been used for the treatment of cancer for over six decades, yet their repertoire continues to grow. These agents act during all phases of the cell cycle, directly on DNA, crosslinking the N-7-guanine residues, causing DNA strand breaks, leading to abnormal base pairing, inhibition of cell division and eventually resulting in cell death. Their systemic toxicity and drug resistance are major obstacles in limiting their clinical efficacy. Major thrust is being laid on discovery of combinations of alkylating agents with other anticancer agents or inhibitors of DNA repair enzymes, topoisomerases, COX-2, p34cdc2 kinase, phosphatases, multi-drug resistance proteins and antivascular agents for improving their clinical efficacy. In addition, novel modulators, such as naphthalimides and their analogues and selective androgen receptor modulators, are being developed to increase the sensitivity of cancer cells to alkylating anticancer agents. This review focuses on patents published during 2005 – 2006 on alkylating anticancer agents.
We report results of a study utilizing a recently developed tissue diagnostic method, based on label-free spectral techniques, for the classification of lung cancer histopathological samples from a tissue microarray. The spectral diagnostic method allows reproducible and objective diagnosis of unstained tissue sections. This is accomplished by acquiring infrared hyperspectral data sets containing thousands of spectra, each collected from tissue pixels about 6 μm on edge; these pixel spectra contain an encoded snapshot of the entire biochemical composition of the pixel area. The hyperspectral data sets are subsequently decoded by methods of multivariate analysis, which reveal changes in the biochemical composition between tissue types, and between various stages and states of disease. In this study, a detailed comparison between classical and spectral histopathology (SHP) is presented, which suggests SHP can achieve levels of diagnostic accuracy that is comparable to that of multi-panel immunohistochemistry.
Human PC-3 prostate cancer cells were incubated in the presence of two cardenolides, i.e., ouabain and 19-hydroxy-2''-oxovoruscharin. Their effects were monitored by infrared spectroscopy of the cells after different exposure times to the cardenolides. Analysis of changes in absorbance intensities indicated that, for both compounds, the absorbance at one wavenumber with a minor contribution of a second wavenumber is sufficient to build a linear model accurate enough to assign more than 97% of the spectra to their correct time slot. Student t-tests and twodimensional correlation analysis (2D-COS) indicated that both drugs have very similar effects on PC-3 cells. However, asynchronous 2D maps revealed significant differences and allowed the sequence of the spectral changes to be determined: 1395 → 1695 cm(-1) for ouabain, and 1400 → 1655 → 1100 → 1250 → 1020 cm(-1) for 19-hydroxy-2''-oxovoruscharin. 2D correlation map subtraction allowed the identification of very specific differences in the impact of both compounds on PC-3 cells, in particular the ability of 19-hydroxy-2''-oxovoruscharin to affect nucleic acid of PC-3 cells.
MicroRNAs (miRNAs), are ~22 nucleotides long, non-coding RNAs that control gene expression post-transcriptionally by binding to their target mRNA's 3'UTRs (untranslated regions). Due to their roles in various important regulatory processes and pathways, miRNAs have been implicated in disease mechanisms such as tumorigenesis when their expression is deregulated. To date, a significant number of miRNAs and their target messenger RNAs (mRNAs) have been identified and verified. It is generally accepted that miRNAs can potentially bind to many mRNAs, which brings the requirement of validation of these interactions. While understanding that such individual interactions is crucial to delineate the role of a specific miRNA, we took a holistic approach and analyzed global changes in the cell due to expression of a miRNA in a model cell line system. Our model consisted of MCF7 cells stably transfected with miR-125b (MCF7-125b) and empty vector (MCF7-EV). MiR-125b is one of the known down-regulated miRNAs in breast cancers. In this study we examined the global structural changes in MCF7 cells lacking and expressing miR-125b by Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) Spectroscopy and investigated the dynamic changes by more sensitive spin-labelling Electron Spin Resonance (ESR) spectroscopy. Our results revealed less RNA, protein, lipid, and glycogen content in MCF7-125b compared to MCF7-EV cells. Membrane fluidity and proliferation rate were shown to be lower in MCF7-125b cells. Based on these changes, MCF7-125b and MCF7-EV cells were discriminated successfully by cluster analysis. Here, we provide a novel means to understand the global effects of miRNAs in cells. Potential applications of this approach are not only limited to research purposes. Such a strategy is also promising to pioneer the development of future diagnostic tools for deregulated miRNA expression in patient samples.
Infrared spectroscopic cytology is potentially a powerful clinical tool. However, in order for it to be successful, practitioners must be able to extract reliably a pure absorption spectrum from a measured spectrum that often contains many confounding factors. The most intractable problem to date is the, so called, dispersion artefact which most prominently manifests itself as a sharp decrease in absorbance on the high wavenumber side of the amide I band in the measured spectrum, exhibiting a derivative-like line shape. In this paper we use synchrotron radiation FTIR micro-spectroscopy to record spectra of mono-dispersed poly(methyl methacrylate) (PMMA) spheres of systematically varying size and demonstrate that the spectral distortions in the data can be understood in terms of resonant Mie scattering. A full understanding of this effect will enable us to develop strategies for deconvolving the scattering contribution and recovering the pure absorption spectrum, thus removing one of the last technological barriers to the development of clinical spectroscopic cytology.
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.
Infrared spectra of single biological cells often exhibit the 'dispersion artefact' observed as a sharp decrease in intensity on the high wavenumber side of absorption bands, in particular the Amide I band at approximately 1655 cm(-1), causing a downward shift of the true peak position. The presence of this effect makes any biochemical interpretation of the spectra unreliable. Recent theory has shed light on the origins of the 'dispersion artefact' which has been attributed to resonant Mie scattering (RMieS). In this paper a preliminary algorithm for correcting RMieS is presented and evaluated using simulated data. Results show that the 'dispersion artefact' appears to be removed; however, the correction is not perfect. An iterative approach was subsequently implemented whereby the reference spectrum is improved after each iteration, resulting in a more accurate correction. Consequently the corrected spectra become increasingly more representative of the pure absorbance spectra. Using this correction method reliable peak positions can be obtained.
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.
Recent results suggest that infrared microspectroscopy, coupled to methods of multivariate analysis, can distinguish normal from virally infected cells. This was established for infection of the oral cavity by the herpes simplex virus, which is associated with the outbreak of cold sores, and for infection of the cervix uteri by the human papillomavirus (HPV). In both cases, subtle (but different) spectral changes are observed that could be used for fast and inexpensive viral screening.
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
Written by the Nobel Prize Laureate in Physiology or Medicine 2008 In the 1970s, the author of this work and his co-workers initially found Epstein-Barr virus DNA in Burkitt's lymphomas and nasopharyngeal cancer and made the connection between HPV infection and cervical cancer. It was also during this period and subsequently that scientists all over the world discovered tumor-inducing bacteria, viruses, parasites, and protozoa, opening up entirely new prospects for the prevention and treatment of infection-induced cancer by vaccination. Adopting a unifying concept and a consistent structure for the individual chapters, Professor zur Hausen provides a thorough and comprehensive overview on cancer-inducing infective agents - viruses, bacteria and parasites - and their corresponding transforming capacities and mechanisms. He does not cover the structure and molecular biology of the agents presented in great detail, but rather concentrates on those aspects that link the respective agents to human oncogenesis. As such, an extensive bibliography after each chapter permits further studies on the subject. With a chapter on Helicobacter written by James Fox and his colleagues at Harvard University, this is an invaluable and instructive reference for all oncologists, microbiologists and molecular biologists working in the area of infections and cancer.
Due to chronic duodeno-gastro-esophageal reflux, the epithelial lining of the esophagus can undergo a change to a more intestinal like type of epithelium. This condition, referred to as Barrett’s esophagus, markedly increases the risk of developing esophageal adenocarcinoma [1]. This type of cancer is rapidly increasing in western society and shows a very high mortality, primarily due to late detection. Early detection of malignancy can significantly increase survival rates. Most detection methods depend on histological examination from endoscopically collected biopsies. However, there are no endoscopically clearly observable differences between healthy tissue, metaplasia and severe displasia. Therefore it is necessary to randomly collect biopsies, with a significant risk of missing malignant degenerations.
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.
Background: Barrett’s oesophagus is the columnar-lined metaplasia that occurs in response to severe gastro-oesophageal reflux and accounts for the dramatic rise in adenocarcinoma at the gastro-oesophageal junction. Diagnostic methods: Endoscopic recognition and pathological diagnosis of the condition is fraught with erroneous interpretation of the pre-malignant degeneration of dysplasia. Screening and surveillance programmes have yet to impact on the disease. Photodiagnosis by spectroscopy and imaging is under intense investigation. The methods can be divided into two groups of morphological (elastic scattering, optical coherence tomography) and molecular and biochemical (Raman and fluorescence). The major diagnostic problem remains the differentiation between inflammation and dysplasia. Raman spectroscopy does offer molecular-specific diagnosis and fibre-optic probes are being developed. The future appears to be multi-modal imaging combined with spectroscopy. Results: Photodynamic therapy is a realistic option for the eradication of dysplastic Barrett’s oesophagus. A recently reported randomised trial has demonstrated a significant improvement in the eradication of dysplasia and prevention of oesophageal cancer. Conclusions: Optical diagnosis and optical eradication have a bright future for the management of Barrett’s oesophagus.
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.
Background: Barrett’s oesophagus is the columnar-lined metaplasia that occurs in response to severe gastro-oesophageal reflux and accounts for the dramatic rise in adenocarcinoma at the gastro-oesophageal junction. Diagnostic methods: Endoscopic recognition and pathological diagnosis of the condition is fraught with erroneous interpretation of the pre-malignant degeneration of dysplasia. Screening and surveillance programmes have yet to impact on the disease. Photodiagnosis by spectroscopy and imaging is under intense investigation. The methods can be divided into two groups of morphological (elastic scattering, optical coherence tomography) and molecular and biochemical (Raman and fluorescence). The major diagnostic problem remains the differentiation between inflammation and dysplasia. Raman spectroscopy does offer molecular-specific diagnosis and fibre-optic probes are being developed. The future appears to be multi-modal imaging combined with spectroscopy. Results: Photodynamic therapy is a realistic option for the eradication of dysplastic Barrett’s oesophagus. A recently reported randomised trial has demonstrated a significant improvement in the eradication of dysplasia and prevention of oesophageal cancer. Conclusions: Optical diagnosis and optical eradication have a bright future for the management of Barrett’s oesophagus.
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.
Infrared spectra of cervical tissue, obtained by biopsy from the squamous-columnar junction, are reported. The spectral patterns observed for columnar tissue are quite different from those of squamous epithelium. Subsequently, the spectra observed for columnar cells in tissue samples were also detected in the spectra of exfoliated cells, indicating the presence of endocervical cells. The columnar or glandular cells exhibit spectral features similar to those observed for pure cervical mucus. © 1997 John Wiley & Sons, Inc. Biospectroscopy 3: 253–257, 1997
It is well known that the infinite Kramers–Kronig transform is equivalent to the infinite Hilbert transform, which is equivalent to the allied Fourier integrals. The Hilbert transform can thus be implemented using fast Fourier transform routines. Such implementation is usually some 60 times faster than the Kramers–Kronig transform for a data file containing about 7 points. This paper reports that, for transformations between the real and imaginary refractive indices, and in , the FFT-based Hilbert transform can be much less accurate than, or as accurate as, the Kramers–Kronig transform, depending on the algorithm used. The Kramers–Kronig transform, incorporating Mclaurin's formula for finding the principal value of the integral, transforms spectra into spectra that are accurate to about 0.05%. Some Hilbert transform algorithms in the literature yield only about 4% accuracy. The BZ algorithm for the Hilbert transform is presented, for use on a laboratory computer running under DOS, that yields spectra accurate to 0.05%. For the transform from to , the BZ algorithm gives accurate to about −0.2% of the largest k value in the spectrum. This compares with an accuracy of 0.5% for the Kramers–Kronig transform. In cases where the spectrum is truncated at low wavenumbers, a simple method is presented that improves by a factor of ~10 the accuracy at low wavenumber of the spectrum obtained by Hilbert or Kramers–Kronig transforms of the spectrum. Keywords: infrared intensities, complex refractive indices, Kramers–Kronig transform, Hilbert transform, optical constants.
This paper presents a short review on the improvements in data processing for spectral cytopathology, the diagnostic method developed for large scale diagnostic analysis of spectral data of individual dried and fixed cells. This review is followed by the analysis of the confounding effects introduced by utilizing reflecting "low-emissivity" (low-e) slides as sample substrates in infrared micro-spectroscopy of biological samples such as individual dried cells or tissue sections. The artifact introduced by these substrates, referred to as the "standing electromagnetic wave" artifact, indeed, distorts the spectra noticeably, as postulated recently by several research groups. An analysis of the standing wave effect reveals that careful data pre-processing can reduce the spurious effects to a level where they are not creating a major problem for spectral cytopathology and spectral histopathology.
This chapter outlines the use of Raman spectroscopy for discrimination of pathologies in the bladder and esophagus based upon the biochemical signature accompanying the disease process. This has great strength in that objective molecular-specific analysis becomes possible for diagnosis and understanding of carcinogenesis processes. The importance of the gold-standard histopathology, sample handling procedures and spectrometer standardization are outlined. Methods of sampling in vivo and in vitro are discussed and discrimination methodologies are demonstrated. Finally exploitation of the inherent biochemical signature found within the tissues is explored and an attempt is made to provide relative concentrations of significant biochemical components.
Keywords:
esophagus;
bladder;
Raman;
biochemistry;
standardization;
histopathology;
discrimination;
cancer;
dysplasia
This paper explores different phenomena that cause distortions of infrared absorption spectra by mixing of reflective and absorptive band shape components of infrared spectra, and the resulting distortion of observed band shapes. In the context of this paper, we refer to the line shape of the variations of the refractive index in spectral regions of an absorption maximum (i.e., in regions of "anomalous dispersion") as "dispersive" or "reflective" line shape contributions, in analogy to previous spectroscopic literature. These distortions usually result in asymmetric bands with a negative intensity contribution at the high wavenumber of the band, accompanied by a shift toward lower wavenumber, and confounded band intensities. In extreme cases of band distortions caused by the "resonance Mie" (RMie) mechanism, spectral peaks may be split into doublets of peaks, change from positive to negative peaks, or appear as derivative-shaped features.
We investigated the hormonal influences on cervical cells using infrared microspectroscopy and found that there were observable spectral changes occurring throughout the cycle. The main differences were seen in the glycogen region (1200–1000 cm−1) and the greatest cyclical variation was observed in spectra of ectocervical cells of women not taking any form of oral contraception. Ectocervical cells from women taking monophasic contraception and endocervical cells from both groups did not display the same degree of variation. Principal component analysis revealed that, although there is cyclical variation, these cells are normal and discrimination between histologically normal and abnormal (high-grade dysplasia) cells was maintained.
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)
IntroductionMethods
Results and DiscussionConclusions
AcknowledgmentsReferences
The use of vibrational spectroscopy in the detection of cancer is a newly emerging diagnostic field, which has shown great potential to date. Many investigations have been carried out on frozen tissue samples, which by their very nature are hard to obtain. However, histology departments have archives of thousands of tissue samples, preserved and mounted in wax blocks. If this archival material can be shown to yield good Raman and IR spectra capable of differentiating between normal and cancerous tissue, it would improve the diagnostic capabilities of spectroscopy even further. Results from these formalin-fixed paraffin processed (FFPP) tissue sections, will give a better understanding of the effects of processing and could unlock the potential diagnostic capabilities of FFPP sections. This study investigated the effect of freezing, formalin fixation, wax embedding and de-waxing. Spectra were recorded from parallel tissue sections of placenta to examine biochemical changes before, during and after processing with both Raman and IR spectroscopy. FFPP sections were shown to provide good quality Raman and IR spectra but new peaks due to freezing and formalin fixation as well as shifts in the amide bands resulting from changes in protein conformation and possible cross-links were found. Residual wax peaks were observed clearly in the Raman spectra. In the FT-IR spectra a single wax contribution was seen which may contaminate the characteristic CH3 deformation band in biological tissue. This study has therefore confirmed that FFPP sections have diagnostic potential provided that researchers are aware of the biochemical changes due to tissue processing highlighted by this study.
A bestselling classic reference, now expanded and updated to cover the latest instrumentation, methods, and applications. The Second Edition of Fourier Transform Infrared Spectrometry brings this core reference up to date on the uses of FT-IR spectrometers today. The book starts with an in-depth description of the theory and current instrumentation of FT-IR spectrometry, with full chapters devoted to signal-to-noise ratio and photometric accuracy. Many diverse types of sampling techniques and data processing routines, most of which can be performed on even the less expensive instruments, are then described. Extensively updated, the Second Edition: Discusses improvements in optical components. Features a full chapter on FT Raman Spectrometry. Contains new chapters that focus on different ways of measuring spectra by FT-IR spectrometry, including fourteen chapters on such techniques as microspectroscopy, internal and external reflection, and emission and photoacoustic spectrometry. Includes a new chapter introducing the theory of vibrational spectrometry. Organizes material according to sampling techniques. Designed to help practitioners using FT-IR capitalize on the plethora of techniques for modern FT-IR spectrometry and plan their experimental procedures correctly, this is a practical, hands-on reference for chemists and analysts. It's also a great resource for students who need to understand the theory, instrumentation, and applications of FT-IR.
We report a computational method to remove or reduce dispersion artifacts from infrared microspectral data collected in transflection (reflection/absorption) mode. This artifact occurs along the edges of tissue samples, in particular if the tissue does not adhere well to the substrate. The method proposed for the removal of the artifact is similar to the phase correction used in standard Fourier transform 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.
Nanotechnology is playing an increasing role in targeted drug delivery into pathological tissues. Drug-loaded pharmaceutical nanocarriers can be delivered into diseased sites by passive targeting (spontaneous accumulation of nanocarriers in the areas with affected vasculature) or by active targeting (via site-specific ligands attached to the surface of drug-loaded nanocarriers). Subsequent level of targeting requires cellular internalization of nanocarriers and their specific association with certain individual cell organelles. The control over intracellular distribution of pharmaceutical nanocarriers requires effective and noninvasive methods of their visualization inside cells. In an attempt to enhance cellular internalization of pharmaceutical nanocarriers and their association with mitochondria specifically, we have prepared three types of cationic liposomes and investigated their intracellular distribution. The analysis was performed using Raman microspectroscopy in order to provide morphological information as well as biochemical signatures of the sample. It was demonstrated that Raman microscopy allows evaluation of the extent of mitochondrial association depending on the liposome composition.
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.
The tumor microenvironment, or stroma, is chemically and morphologically modified during carcinoma progression. The predominant cell type in the stroma, the fibroblast, maintains collagen properties in normal tissue and often transformed during tumor progression. Biochemical changes within fibroblasts upon initial cancer activation, however, are relatively poorly defined. Here, we hypothesized that Fourier transform infrared (FT-IR) spectroscopic imaging could potentially be employed to examine these early transformations. Further, we employ attenuated total reflectance (ATR) microscopy to characterize subcellular spectra and their changes upon transformation. We characterized fibroblast transitions upon stimulation with both a molecular agent and a carcinoma-mimicking cellular co-culture system. Changes were predominantly observed in the 1080 cm(-1) and 1224 cm(-1) peak absorbance, commonly associated with nucleic acids, as well as in the band at 2930 cm(-1) associated with the C-H stretching of proteins in the cytoplasmic compartment. In conclusion, biochemical changes in cancer-associated fibroblasts that express α-SMA are dominated by the cytoplasm, rather than the nucleus. This ensures that spectral changes are not associated with proliferation or cell cycle processes of the cells and the cells are undergoing a true phenotypic change denoted by protein modifications in the cell body.
The present study investigated the impact of nano titanium dioxide (nTiO 2) exposure on the cellular structures of the nitrogen-fixing cyanobacteria Anabaena variabilis. Results of the present study showed that nTiO 2 exposure led to observable alteration in various intracellular structures and induced a series of recognized stress responses, including production of reactive oxygen species (ROS), appearance and increase in the abundance of membrane crystalline inclusions, membrane mucilage layer formation, opening of intrathylakoidal spaces, and internal plasma membrane disruption. The production of total ROS in A. variabilis cells increased with increasing nTiO 2 doses and exposure time, and the intracellular ROS contributed to only a small fraction (<10%) of the total ROS measured. The percentage of cells with loss of thylakoids and growth of membrane crystalline inclusions increased as the nTiO 2 dose and exposure time increased compared with controls, suggesting their possible roles in stress response to nTiO 2, as previously shown for metals. Algal cell surface morphology and mechanical properties were modified by nTiO 2 exposure, as indicated by the increase in cell surface roughness and shifts in cell spring constant determined by atomic force microscopy analysis. The change in cell surface structure and increase in the cellular turgor pressure likely resulted from the structural membrane damage mediated by the ROS production. Transmission electron microscopy (TEM) analysis of nTiO 2 aggregates size distribution seems to suggest possible disaggregation of nTiO 2 aggregates when in close contact with microbial cells, potentially as a result of biomolecules such as DNA excreted by organisms that may serve as a biodispersant. The present study also showed, for the first time, with both TEM and Raman imaging that internalization of nTiO 2 particles through multilayered membranes in algal cells is possible.
Recently a new di-gold(I) organometallic complex [1,3-(Ph(3)PAu)(2)-C(6)H(4)] (KF0101) has been synthesised and found to exhibit cytotoxic activity in vitro. Subsequently it has been demonstrated that KF0101 shows little or no cross-resistance against a number of the cisplatin resistant ovarian cancer cell lines in vitro suggesting a different mode of action for the drug. In this study, syncrotron radiation infrared microspectroscopy (SR-IRMS) has been used on drug treated single A2780 cells in order to determine if this different mode of action can be identified spectroscopically. The aim of the study was to establish: (i) if single cell SR-IRMS could be used to give insight into the cellular response on treatment with different cytotoxic agents relative to non-treated cells (control) and (ii) that if the cytotoxic drugs elicit a different biochemical response these responses could be distinguished from each other. The most striking features obtained after Principal Components Analysis (PCA) of Resonant Mie Scattering (RMieS) corrected single cell spectra of drug treated ovarian A2780 cells are: (i) The spectra obtained for the control are quite heterogeneous and several hundred spectra are required to adequately define the nature of the control; (ii) after drug treatment at the IC50 level for 24 h with cisplatin, KF0101, methotrexate, paclitaxel or 5-fluorouracil the cell spectra, as represented on a PCA scores plot, generally concentrate in certain well defined areas of the control, there are however a small number of spectra that fall outside of the area defined by the control; and (iii) a differentiation between cell spectra obtained on treatment with different drugs is observed which fits well with different in vitro cell culture behaviour and a flow cytometry cell cycle analysis of the control and drug treated cells. Inspection of the loading plots shows that PC1 is essentially the same for all plots and reflects changes in cell biochemistry related to the cell cycle. PC2, however, on comparison of the control versus cisplatin or cisplatin versus KF0101 is indicative of differences induced by drug treatment and has been termed as cell cycle-plus behaviour. These data are shown to be consistent with that obtained using bench-top IRMS by averaging a number of single cell spectra and carrying out a PCA, but SR-IRMS offers more insight into how the drug is affecting the cell population. More importantly, this approach enables the influence of the cell cycle on both the control and drug treated samples to be taken into consideration when evaluating the drug-cell interaction.
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.
Early detection of (pre-)cancerous changes improves prognosis, therefore in the UK patients at high risk of developing gastrointestinal cancers are enrolled on endoscopic surveillance programmes or the Bowel Cancer Screening Programme. The current gold standard technique for the detection of pre-cancerous changes in the gastrointestinal tract is histopathological analysis of biopsy tissue collected at endoscopy. This relies upon subjective assessment of morphological changes within the excised tissue samples and poor targeting of pre-malignant lesions. Raman spectroscopy offers a number of potential advantages for in vivo assessment of tissue at endoscopy. The performance of a custom built Raman probe as a biopsy targeting tool has been evaluated using excised biopsy material. Multivariate classification models have been used to demonstrate the likely ability of a miniature, confocal, fibre optic Raman probe to be used as an optical biopsy tool at endoscopy to provide spectral information in clinically practicable timescales. This technique could facilitate improved targeting of excisional biopsy with associated clinical benefits.
Confocal Raman micro-spectral imaging (CRMI) is a relatively novel technique for the construction of label-free images of biological entities, such as cells or tissue sections. This method utilizes thousands of spatially resolved Raman spectra, and sophisticated image analysis algorithms, to construct images which are based strictly on the inherent biochemical abundance contrast afforded by Raman microscopy. Here, we apply this methodology to monitor the very early communication processes that occur in a growing stem cell colony, yielding information on biochemical composition during development processes of the early stages of embryogenesis.
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.
In this manuscript, we report the application of EMSC to correct infrared micro-spectral data recorded from tissue that describe resonant Mie scattering contributions. Small breast micro-metastases previously undetectable using the raw measured spectra were provided clear contrast from the surrounding tissue after signal correction. The technique also proved transferrable, successfully correcting imaging data sets recorded from multiple patients. It is envisaged more robust methods of supervised analysis can now be constructed to automatically classify and diagnose tissue spectra.
Fourier transform infrared (FT-IR) spectroscopic imaging combines the specificity of optical microscopy with the spectral selectivity of vibrational spectroscopy. There is increasing recognition that the recorded data may be dependent on the optical configuration and sample morphology in addition to its local material spectral response, but a quantitative framework for predicting such dependence is lacking. Here, a theory is developed to relate recorded data to the spectral and physical properties of heterogeneous samples. The modeling approach combines optical theory through rigorous coupled wave analysis with modeling of sampling geometry and sample structure. The interplay of morphology and dispersion are systematically explored using increasingly sophisticated samples to illustrate the dependence of the detected optical intensity on the spatial sample structure. Predictions of spectral distortions arising from the sample structure are quantified, and experimental validation of the developed theory is performed using a microfabricated standard from a commercial photoresist polymer. The developed framework forms a basis for understanding sample induced distortions in spectroscopic IR microscopy and imaging.
Midinfrared (IR) microspectroscopy is widely employed for spatially localized spectral analyses. A comprehensive theoretical model for the technique, however, has not been previously proposed. In this paper, rigorous theory is presented for IR absorption microspectroscopy by using Maxwell's equations to model beam propagation. Focusing effects, material dispersion, and the geometry of the sample are accounted to predict spectral response for homogeneous samples. Predictions are validated experimentally using Fourier transform IR (FT-IR) microspectroscopic examination of a photoresist. The results emphasize that meaningful interpretation of IR microspectroscopic data must involve an understanding of the coupled optical effects associated with the sample, substrate properties, and microscopy configuration. Simulations provide guidance for developing experimental methods and future instrument design by quantifying distortions in the recorded data. Distortions are especially severe for transflection mode and for samples mounted on certain substrates. Last, the model generalizes to rigorously consider the effects of focusing. While spectral analyses range from examining gross spectral features to assessing subtle features using advanced chemometrics, the limitations imposed by these effects in the data acquisition on the information available are less clear. The distorting effects are shown to be larger than noise levels seen in modern spectrometers. Hence, the model provides a framework to quantify spectral distortions that may limit the accuracy of information or present confounding effects in microspectroscopy.
Cyclophosphamide (CPA) is a DNA alkylating agent widely used in cancer chemotherapy. CPA undergoes metabolic activation to phosphoramide mustard and nornitrogen mustard (NOR) which alkylate the N-7 position of guanine in DNA to produce N-[2-(N7-guaninyl) ethyl]-N-[2-hydroxyethyl]-amine (G-NOR-OH) monoadducts and N,N-bis[2-(N7-guaninyl) ethyl] amine cross-links (G-NOR-G). G-NOR-G cross-links are strongly cytotoxic and are thought to be responsible for the biological activity of CPA. In the present work, an isotope dilution high-performance liquid chromatography-electrospray ionization (positive ion) tandem mass spectrometry (HPLC-ESI(+)-MS/MS) methodology was developed to accurately quantify G-NOR-G adducts in human blood. In our approach, DNA extracted from white blood cells (5-20 microg) is spiked with an internal standard of [(15)N(10)]-G-NOR-G and subjected to thermal hydrolysis to release G-NOR-G adducts from the DNA backbone. Following solid phase extraction, G-NOR-G conjugates are quantified by capillary HPLC-ESI-MS/MS in the selected reaction monitoring mode. The application of the new methodology is demonstrated for DNA extracted from blood of three cancer patients receiving 50-60 mg/kg of intravenous CPA. The highest numbers of G-NOR-G adduct (up to 18 adducts per 10(6) normal nucleotides) were observed 4-8 h following CPA administration, followed by a gradual decrease over time, probably due to adduct hydrolysis, DNA repair, and white blood cell death. This methodology will be useful for future investigations of the interindividual differences for CPA-induced DNA-DNA cross-linking.