Abstract Aggregation‐induced emission (AIE) luminogens are promising tools for biological imaging, yet their utility is often limited by nonspecific aggregation and elevated background fluorescence. Here, we present a bioorthogonal in situ formation strategy that enables precise, organelle‐specific activation of AIEgens within live cells. This system employs a dual‐lock mechanism—combining tetrazine quenching and twisted intramolecular charge shuttle (TICS) dynamics—to maintain an ultra‐low fluorescence background in the precursor state, even under aggregating conditions. Upon bioorthogonal reactions and molecular aggregation, the resulting AIEgens exhibit tunable emission peaks ranging from 605 to 665 nm, large Stokes shifts (up to 201 nm), and an exceptional fluorescence enhancement (up to 1033‐fold). Their high biocompatibility and spatial precision allow for multiplexed, simultaneous labeling of intracellular targets. Compared to conventional fluorogenic and bioorthogonal probes, these AIEgens exhibit a sigmoidal amplification response, enabling the sensitive discrimination of subtle biomarker expression differences and the effective identification of injured cells during disease progression. This strategy significantly enhances the specificity and sensitivity of live‐cell imaging, expands the functional utility of AIE luminogens, and offers a versatile platform for high‐resolution, multiplexed bioimaging in biomedical research and diagnostics.
Yu et al. (Thu,) studied this question.