Raman spectral imaging of single cancer cells: probing the impact of sample fixation methods |
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Authors: | Florence Draux Cyril Gobinet Josep Sulé-Suso Aurélie Trussardi Michel Manfait Pierre Jeannesson Ganesh D Sockalingum |
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Institution: | 1. Unité MéDIAN, Université de Reims Champagne-Ardenne, CNRS UMR 6237–MEDyC, UFR de Pharmacie, IFR53, 51 rue Cognacq-Jay, 51096, Reims Cedex, France 2. Institute for Science and Technology in Medicine, Guy Hilton Research Centre, Keele University, Thornburrow Drive, Stoke-on-Trent, ST4 7QB, UK
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Abstract: | Raman spectroscopy has proven its potential for the analysis of cell constituents and processes. However, sample preparation
methods compatible with clinical practice must be implemented for collection of accurate spectral information. This study
aims at assessing, using micro-Raman imaging, the effects of some routinely used fixation methods such as formalin-fixation,
formalin-fixation/air drying, cytocentrifugation, and air drying on intracellular spectral information. Data were compared
with those acquired from single living cells. In parallel to these spectral information, cell morphological modifications
that accompany sample preparation were compared. Spectral images of isolated cells were first analyzed in an unsupervised
way using hierarchical cluster analysis (HCA), which allowed delimitation of the cellular compartments. The resulting nuclei
cluster centers were compared and revealed at the molecular level that fixation induced changes in spectral information assigned
to nucleic acids and proteins. In a second approach, a supervised fitting procedure using model spectra of DNA, RNA, and proteins,
chemically extracted from living cells, revealed very small modifications at the level of the localization and quantification
of these macromolecules. Finally, HCA and principal components analysis (PCA) performed on individual spectra randomly selected
from the nuclear regions showed that formalin-fixation and cytocentrifugation are sample preparation methods that have little
impact on the biochemical information as compared to living conditions. Any step involving cell air drying seems to accentuate
the spectral deviations from the other preparation methods. It is therefore important in a future context of spectral cytology
to take into account these variations. |
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