Multimodal measurements of phototoxicity in nonlinear optical microscopy

Nonlinear optical imaging modalities, such as two-photon microscopy and stimulated Raman scattering (SRS) microscopy, make use of pulsed-laser excitation with high peak intensity that can perturb the native state of cells. In this study, we investigated the short and long-term effects of pulsed laser induced phototoxicity. We used bulk RNA sequencing, quantitative measurement of cell proliferation, and measurement of the generation of reactive oxygen species (ROS) to assess phototoxic effects, at different time scales, for a range of laser excitation settings relevant to SRS imaging. We define a range of laser excitation settings for which there was no significant ROS generation, differential gene expression, or change in proliferation rates of mouse Neuro2A cells. Changes in proliferation rate and ROS generation were observed under imaging conditions with an excitation intensity of over 600 mW/m2. Repeated imaging of the same field of view at this excitation intensity of over 600 mW/m2 resulted in visual damage to N2A cells. Laser induced perturbations in live cells may impact downstream measurements of cell state including subsequent imaging or molecular measurements. 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.