A blue light-activatable coumarin-tetrazine probe for lipid droplet imaging.

Despite the prevalent use of 1,2,4,5-tetrazine (Tz) as a fluorescence quencher in fluorogenic probes, whose fluorescence can be restored via bioorthogonal reactions with complementary dienophiles for labeling or imaging applications, photoactivation has emerged as a powerful alternative for precise spatiotemporal control. Notwithstanding this advantage, existing coumarin-tetrazine probes are hindered by suboptimal photochemical efficiency and high activation energies. Herein, we designed coumarin-tetrazine conjugates linked via a vinyl spacer at the C3 position of coumarin that are efficiently activatable by blue light. This process generates superoxide anions to destroy the tetrazine moiety, thereby restoring the coumarin fluorescence. This finding illuminates a new facet of tetrazine reactivity and opens avenues for advanced fluorogenic probe design. As a proof of concept, we developed a coumarin-tetrazine probe targeting lipid droplets based on this strategy, allowing high-resolution visualization of these organelles in living cells. This work not only uncovers a new reactivity mode of tetrazine but also provides a versatile platform for designing photoactivatable fluorogenic probes for live-cell imaging.