Phototoxic effects of nonlinear optical microscopy on cell cycle, oxidative states, and gene expression

Nonlinear optical imaging modalities, such as stimulated Raman scattering (SRS) microscopy, use pulsed-laser excitation with high peak intensity that can perturb the native state of cells. In this study, we used bulk RNA sequencing, quantitative measurement of cell proliferation, and fluorescent measurement of the generation of reactive oxygen species to assess phototoxic effects of near-IR pulsed laser radiation, at different time scales, for laser excitation settings relevant to SRS imaging. We define a range of laser excitation settings for which there was no significant change in mouse Neuro2A cells after laser exposure. This study provides guidance for imaging parameters that minimize photo-induced perturbations in SRS microscopy to ensure accurate interpretation of experiments with time-lapse imaging or with paired measurements of imaging and sequencing on the same cells.