No abstract

Raman microspectroscopy allows probing subcellular compartments and provides a unique spectral fingerprint indicative of endogenous molecular composition. Although several spectroscopic cell studies have been reported on fixed samples, only few attempts concern single growing cells. Here, we have tested different optical substrates that would best preserve cell integrity and allow direct measurement of Raman spectra at the single living cell level. Calu-1 lung cancer cells were used as a model and their morphology and growth were assessed on Raman substrates like quartz, calcium fluoride, and zinc selenide. Data show that quartz was the most appropriate taking into consideration both cell morphology and proliferation rate (47% on quartz vs. 55% of BrdU-positive cells on conventional plastic). Using quartz, 40 cells were analysed and Raman spectra were collected from nuclei and cytoplasms using a 785 nm laser excitation of 30 mW at the sample, in the spectral range of 580-1750 cm(-1), and an acquisition time of 2 x 10 sec/spectrum. Discriminant spectral information related to nucleus and cytoplasm were extracted by multivariate statistical methods and attributed to nucleic acids, lipids, and proteins. Finally, Raman spectral imaging was performed to show the distribution of these components within the cell.

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.