Diem’s scientific contributions

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