Photoactivatable fluorophores enable selective fluorescence activation within a defined region of interest at a precisely controlled time point. This high degree of spatiotemporal control over fluorescence emission facilitates advanced imaging modalities and super-resolution microscopy techniques, allowing real-time monitoring of dynamic processes and nanoscale visualization of structural features. The development of photocage groups is the general and most popular strategy for designing photoactivatable fluorophores. Herein, we present the hydroxy-pendant phenyl tetrazole as a novel photocage for push-pull photoactivatable fluorophores. Upon photolysis, this tetrazole-based photocage undergoes rapid cleavage with nitrogen extrusion, followed by nitrile imine-mediated nucleophilic addition, and subsequent proton transfer to afford the corresponding hydrazone photoproduct. This tetrazole to hydrazone photoconversion leads to an electronic inversion from electron-withdrawing to electron-rich character, thereby triggering the fluorescence turn-on via charge recombination. This phenyl tetrazole photocage was applicable in coumarin (Cou), nitrobenzoxadiazole (NBD), and naphthalimide (NP) fluorophores. The NP-based photoactivatable fluorophores enabled spatiotemporally resolved, targeted live-cell fluorescence imaging in both confocal and stimulated emission depletion (STED) microscopy, exhibiting rapid photoresponse, non-toxic byproduct release, bright fluorescence emission, and a high signal-to-background ratio.
