Optimized two-color super resolution imaging of Drp1 during mitochondrial fission with a slow-switching Dronpa variant

Significance Optimal performance of super resolution fluorescence localization microscopy relies on a clear understanding of the photo-physical properties of photoactivatable (photo-switchable) fluorescent proteins [PA(PS)-FPs] at the single-molecule level. Our comparative study of Dronpa and a novel variant, rsKame, demonstrates the crucial role of photo-switching kinetics in super resolution imaging. rsKame, with its superior properties, significantly broadens the green PA(PS)-FP palette. We demonstrate the efficacy of rsKame and our two-color super resolution imaging method (paired with PAmCherry1) by visualizing the inner and outer mitochondrial membranes and in situ structural parameters of dynamin related protein 1 helical rings during mitochondrial fission. Our two-color super resolution imaging method presented here is a reliable and user-friendly technique without complicated sample preparation.