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.

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 super-imposable. 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.

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.

No abstract