Photobleaching and phototoxicity of mitochondria in live cell fluorescent super-resolution microscopy

Photobleaching and phototoxicity can induce detrimental effects on cell viability and compromise the integrity of collected data, particularly in studies utilizing super-resolution microscopes. Given the involvement of multiple factors, it is currently challenging to propose a single set of standards for assessing the potential of phototoxicity. The objective of this paper is to present empirical data on the effects of photobleaching and phototoxicity on mitochondria during super-resolution imaging of mitochondrial structure and function using Airyscan and the fluorescent structure dyes Mitotracker green (MTG), 10-N-nonyl acridine orange (NAO), and voltage dye Tetramethylrhodamine, Ethyl Ester (TMRE). We discern two related phenomena. First, phototoxicity causes a transformation of mitochondria from tubular to spherical shape, accompanied by a reduction in the number of cristae. Second, phototoxicity impacts the mitochondrial membrane potential. Through these parameters, we discovered that upon illumination, NAO is much more phototoxic to mitochondria compared to MTG or TMRE and that these parameters can be used to evaluate the relative phototoxicity of various mitochondrial dye-illumination combinations during mitochondrial imaging.