Ellen J Marcsisin’s research while affiliated with Northeastern University and other places

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.

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

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.

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.