Ultrafluorogenic mitochondrial probe platform enabled by multichannel barrierless quenching

Abstract Fluorescence imaging enables noninvasive visualization of mitochondrial dynamics, yet conventional fluorogenic probes are often compromised by high background fluorescence and limited target specificity. Herein, we report a molecular scaffold based on multichannel barrierless quenching (MCBQ), which suppresses background emission through barrierless transitions into dual twisted intramolecular charge transfer (TICT) states enabled by flexible pyridinium regioisomer design. Notably, the meta-position pyridinium configuration disrupts resonance delocalization, enhances charge separation, and drives ultrafast MCBQ-mediated quenching within 0.30 ps. Leveraging this MCBQ strategy, we developed a mitochondrial-targeted, ultralow-background fluorogenic probe (MBFP) platform that undergoes biochemical factor-triggered self-immolation to release a bright fluorophore, resulting in up to 2331-fold fluorescence enhancement. As a proof of concept, the H2O2-responsive MBFP enabled selective quantification of mitochondrial H2O2 variations in cellular and murine models, further supporting its translational potential in clinical serum samples. This work establishes MCBQ as a broadly applicable design strategy for ultralow-background mitochondrial fluorogenic probes and provides a versatile platform for real-time monitoring of mitochondrial dysfunction toward early disease diagnosis.