Saline dry fixation for improved cell composition analysis using Raman spectroscopy

• Published in: Analyst (London) 2023-06, Vol.148 (12), p.2745-2757
• Main Authors: Rangan, Shreyas, Wong, Riley, Schulze, H. Georg, Vardaki, Martha Z, Blades, Michael W, Turner, Robin F. B, Piret, James M
• Format: Article
• Language: English
• Subjects: Biomolecules; Composition; Fixation; Formaldehyde; Methanol; Raman spectra; Raman spectroscopy; Spectroscopic analysis; Spectrum analysis; Stability analysis
• ISSN: 0003-2654; 1364-5528
• DOI: 10.1039/d2an01916g

Raman spectroscopy enables the label-free assessment of cellular composition. While live cell analysis is the most accurate approach for cellular Raman spectroscopy, the analysis of fixed cells has proved to be very useful, particularly in collaborative projects where samples need to be serially examined by different laboratories or stored and reanalyzed at a later date. However, many chemicals that are widely used for cell fixation directly affect cellular biomolecules, yielding Raman spectra with missing or altered information. In this article, we compared the suitability of dry-fixation with saline versus chemical fixatives. We compared the Raman spectroscopy of saline dry-fixed cells with the more commonly used formaldehyde and methanol fixation and found that dry-fixed cell spectra preserved more cellular information than either chemical fixative. We also assessed the stability of dry-fixed cells over time and found that they were stable for at least 5 months. Finally, a comparison of dry-fixed and live cell spectra revealed effects due to the hydration state of the cells since they were recovered upon rehydrating dry-fixed samples. Thus, for fixed cell Raman spectroscopy, we recommend dry-fixation with unbuffered saline as a superior method to formaldehyde or methanol fixation. Principal Component Analysis (PCA) of primary human T-cell spectra showed clear separation of different fixatives. Saline dry-fixed cell spectra showed better preservation of donor-to donor variability and improved retention of Raman band shapes.