Long‐Term, Single‐Molecule Imaging of Proteins in Live Cells with Photoregulated Fluxional Fluorophores

Single‐molecule localization microscopy (SMLM) can reveal nanometric details of biological samples, but its high phototoxicity hampers long‐term imaging in live specimens. A significant part of this phototoxicity stems from repeated irradiations that are necessary for controlled switching of fluorophores to maintain the sparse labeling of the sample. Lower phototoxicity can be obtained using fluorophores that blink spontaneously, but controlling the density of single‐molecule emitters is challenging. We recently developed photoregulated fluxional fluorophores (PFFs) that combine the benefits of spontaneously blinking dyes with photocontrol of emitter density. These dyes, however, were limited to imaging acidic organelles in live cells. Herein, we report a systematic study of PFFs that culminates in probes that are functional at physiological pH and operate at longer wavelengths than their predecessors. Moreover, these probes are compatible with HaloTag labeling, thus enabling timelapse, single‐molecule imaging of specific protein targets for exceptionally long times.