Photoswitching Reagent for Super‐Resolution Fluorescence Microscopy

Single‐molecule localization microscopy (SMLM) has revolutionized optical microscopy by exceeding the diffraction limit and revealing previously unattainable nanoscale details of cellular structures and molecular dynamics. This super‐resolution imaging capability relies on fluorophore photoswitching, which is crucial for optimizing the imaging conditions and accurately determining the fluorophore positions. To understand the general on and off photoswitching mechanisms of single dye molecules, various photoswitching reagents were evaluated. Systematic measurement of the single‐molecule‐level fluorescence on and off rates (kon and koff) in the presence of various photoswitching reagents and theoretical calculation of the structure of the photoswitching reagent‐fluorophore pair indicated that the switch‐off mechanism is mainly determined by the nucleophilicity of the photoswitching reagent, and the switch‐on mechanism is a two‐photon‐induced dissociation process, which is related to the power of the illuminating laser and bond dissociation energy of this pair. This study contributes to a broader understanding of the molecular photoswitching mechanism in SMLM imaging and provides a basis for designing improved photoswitching reagents with potential applications extending to materials science and chemistry. In this study, we propose a unified mechanism for photoswitching in super‐resolution fluorescence imaging by analyzing various photoswitching reagents. We found that the switch‐off mechanism is mainly determined by the nucleophilicity of the photoswitching reagent, and the switch‐on mechanism is a two‐photon‐induced dissociation process. Based on this systematic investigation, sodium azide is suggested as a new photoswitching reagent.