Studies of chemical fixation effects in human cell lines using Raman microspectroscopy

• Published in: Analytical and bioanalytical chemistry Vol. 396; no. 5; pp. 1781 - 1791
• Main Authors: Meade, Aidan D, Clarke, Colin, Draux, Florence, Sockalingum, Ganesh D, Manfait, Michel, Lyng, Fiona M, Byrne, Hugh J
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Berlin/Heidelberg : Springer-Verlag 01-03-2010; Springer-Verlag; Springer; Springer Verlag
• Subjects: Acetic acid; Acetic Acid - chemistry; Analysis; Analytical Chemistry; Biochemistry; Carnoy's fixative; cell culture; Cells; Cells, Cultured; Characterization and Evaluation of Materials; Chemical fixation mechanisms; Chemistry; Chemistry and Materials Science; Denaturation; Exact sciences and technology; Fixation; Food Science; Formaldehyde; Formaldehyde - chemistry; Formalin; HCA; Human; Humans; k-means clustering; Laboratory Medicine; Life Sciences; Mathematical analysis; Methanol - chemistry; Methanol-acetic acid; Micro-Raman spectroscopy; Molecular Structure; Monitoring/Environmental Analysis; Multivariate Analysis; Organic acids; Original Paper; PCA; Proteins; Raman spectroscopy; Skin; Skin care products; Spectrometric and optical methods; Spectrum Analysis, Raman; Tissue Fixation - methods; Cell culture; means clustering; Methanol–acetic acid; Carnoy’s fixative; HCA; Formalin; Chemical fixation mechanisms; Micro-Raman spectroscopy; PCA; Lung; Lipids; Time; Formaldehyde; Vibrational spectroscopy; Characterization; Degradation; k-means clustering; Raman spectrometry; Nucleic acid; Methanol; Human; Methanol-acetic acid; Denaturation; Use; Epithelium; In vitro; Leaching; Protein; Non destructive method; Fixation; Cell line; Chemical fixation mechanisms Cell culture; Skin; Technique; Acetic acid; Carnoy's fixative; Principal component analysis; Integrity
• ISSN: 1618-2642; 1618-2650
• 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) employing 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 produces spectral content that is closest to that in the live cell and by extension, best preserves the cellular integrity. 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 and dependent on both the cell line and fixative employed. Moreover, important spectral changes occur with all fixatives that have consequences for the interpretation of biochemical processes within fixed cells. The study further demonstrates the potential of vibrational spectroscopy in the characterization of complex biochemical processes in cells at a molecular level. [graphic removed]