Studies of Chemical Fixation Effects in Human Cell Lines Using Raman Microspectroscopy

Aidan D. Meade, Dublin Institute of Technology
Colin Clarke, Dublin Institute of Technology
Florence Draux, Universite de Reims Champagne-Ardenne
Ganesh Sockalingum, Universite de Reims Champagne-Ardenne
Michel Manfait, Universite de Reims Champagne-Ardenne
Fiona M. Lyng, Dublin Institute of Technology
Hugh J. Byrne, Dublin Institute of Technology

Document Type Article

Analytical and Bioanalytical Chemistry, Volume 396, Number 5 / March, 2010, pp.1781-1791 DOI: 10.1007/s00216-009-3411-7


The in-vitro study of cellular species using Raman spectroscopy has proven a powerful non-invasive modality for the analysis of cell constituents and processes. This work uses micro-Raman spectroscopy to study the chemical fixation mechanism in three human cell lines (normal skin, normal bronchial epithelium, and lung adenocarcinoma) in the presence of fixatives that preferentially preserve proteins (formalin), and nucleic acids (Carnoy’s fixative and Methanol-Acetic Acid). Spectral differences between the mean live cell spectra and fixed cell spectra together with principal components analysis (PCA), and clustering techniques were used to analyse and interpret the spectral changes. The results indicate that fixation in formalin or Carnoy’s fixative preserves the biochemical composition of the cell close to that in the live cell for each of the studied cell lines. Nucleic acid degradation, protein denaturation, and lipid leaching were observed with all fixatives and for all cell lines, but to varying degrees. The results presented here suggest that the mechanism of fixation for short fixation times is complex aUUnd more cell-lidependent than previously observed. Moreover, important spectral changes occur with all fixatives that have consequences for the interpretation of experiments on biochemical processes within fixed cells. 