Effects of Chemical Fixation on Stimulated Raman Scattering Microscopy.

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of cells and tissues in their native state with chemical specificity. However, there are often experimental advantages of chemical fixation of samples prior to imaging, which can introduce perturbations that may alter the native state of the samples and possibly impact the SRS signal. In this study, we systematically characterize the effects of common fixatives (paraformaldehyde, formalin, glutaraldehyde, methanol, ethanol) on the preservation of cellular integrity and molecular composition in a HeLa cell model as observed by SRS microscopy. We demonstrate how the different fixatives can influence lipid and protein content and overall cell morphology, with significant implications for the accuracy of quantitative SRS microscopy. Our findings indicate that formaldehyde-based fixatives impose minimal disruption to the spatial or spectral features of biomolecules compared to the other fixatives tested and suggest glutaraldehyde (GA) as a suitable alternative for SRS imaging. This study provides insights into the choice of optimal sample preparation, enabling more reliable SRS-based studies for the evaluation of cellular processes and disease mechanisms where fixation is used.