The impact of cell fixation on coherent anti-stokes Raman scattering signal intensity in neuronal and glial cell lines.

A number of studies require sample fixation, aimed to preserve cells in a physiological state with minimal changes of morphology and intracellular molecular content. Sample fixation may significantly distort experimental data, which makes the data interpretation process more challenging. It is particularly important for study of lipid-related diseases, where the biochemical and morphological characteristics of the cells need to be well preserved for an accurate data analysis. This study investigates the effects of formaldehyde and ethanol (EtOH) fixatives on coherent anti-stokes Raman scattering (CARS) signal of proteins and lipids in major cellular compartments of neuronal and glial cells. We found that both fixatives induce alteration of proteins and lipids signal in studied cell lines. Furthermore, the impact of sample preservation methods on CARS signal varies between cell lines. For instance, our data reveals that EtOH fixation induces ~45% increase of CARS signal of proteins in the nucleolus of neuronal cells and ~35% decrease of CARS signal in glial cells. The results indicate that aldehyde fixation is a preferable method for preservation of neuronal and glial cells prior to CARS imaging, as it less affects both CARS signal and intracellular distribution of proteins and lipids.