A Tetrazine-Based Light-Assisted Activation Probe for Time-Controllable Monitoring of O₂^•–

Photoactive probes are important derivatives of small molecule fluorescent probes that provide spatiotemporally controllable imaging capabilities. However, conventional photoactive probes are limited by short auxiliary light wavelengths, prolonged irradiation times, and lack of analytes, making it difficult to achieve time-controlled imaging applications. Herein, a tetrazine-based probe (<b>SWJT</b>-<b>28</b>) was synthesized for the light-assisted activation study. Based on the intrinsic photolabile and oxidative properties of tetrazine, <b>SWJT</b>-<b>28</b> could quickly react with the superoxide anion (O₂^•-) and emit fluorescence under blue light irradiation. Unlike the traditional relay recognition strategy, <b>SWJT</b>-<b>28</b> requires the simultaneous presence of both blue light and O₂^•- to trigger fluorescence emission, thereby enabling a one-step activation process and significantly reducing the irradiation and response time (<40 s). Furthermore, <b>SWJT</b>-<b>28</b> demonstrated a high selectivity and excellent stability. This probe was successfully applied for time-controlled imaging in HeLa cells and oxygen-glucose deprivation models, validating the utility of <b>SWJT</b>-<b>28</b> in redox studies.