All-Visible-Light Activated Diarylethene Photoresponsive Probe for Lysosomal STORM Imaging

Stochastic optical reconstruction microscopy (STORM) breaks through the diffraction limit of conventional optical imaging, enabling visualization of biological processes with nanoscale precision. Diarylethene derivatives have attracted particular attention due to their remarkable reversibility, excellent thermal stability, and fatigue resistance. Photoswitchable diarylethene compounds with easy modification and tunable fluorescence eliminate the need for additives in single-molecule localization techniques, opening new development opportunities in STORM imaging. However, most diarylethene probes currently used for STORM imaging typically require a phototoxic ultraviolet (UV) laser to regulate the transition between fluorescent and dark states, and they lack organelle recognition capability. Herein, we propose a novel design strategy for STORM fluorescent probes. By conjugating an intramolecular proton transfer (IPT) fluorophore on one side and a lysosomal targeting group on the other with diarylethene, we successfully synthesized the molecule HMN. This probe not only regulates fluorescence blinking between bright and dark states using harmless all-visible light but also exhibits excellent lysosome-targeting capability, which facilitates intracellular STORM imaging of lysosomes. HMN demonstrates outstanding fatigue resistance, sensitive fluorescence switching, and accurate lysosome recognition. Its fluorescence can be activated by a 488 nm laser and deactivated by a 561 nm laser, without requiring any additives in the imaging medium. Using the photoresponsive probe HMN, we achieved all-visible-light STORM imaging, which helps determine the subcellular distribution of lysosomes and enables super-resolution imaging of individual lysosomes with a resolution of up to 99 nm.