Abstract
Mitochondrial dysfunction and defective mitophagy are defining features of numerous neurodegenerative and metabolic disorders, yet existing tools provide limited ability to quantify mitophagy dynamics in real time within living, post-mitotic cells. Here we present MitoFlare, a mitochondria-targeted, reversible mtROS-responsive fluorogenic probe that enables continuous, non-genetic visualization of mitochondrial oxidative activation and turnover. MitoFlare incorporates dual TEMPO nitroxide quenchers into a long-wavelength rhodamine scaffold, producing >95% basal quenching and rapid, fully reversible fluorescence activation in response to mitochondrial superoxide, hydroxyl radicals, lipid-derived peroxyl species, and peroxynitrite. When combined with LysoTracker Green, MitoFlare forms a dual-probe imaging platform that resolves the entire mitophagy cascade with high spatial and temporal fidelity in intact PC12 neuronal cells. Using this platform, we established a quantitative framework comprising three mechanistically distinct metrics: (i) a proximity index that reports early mitochondrial engagement with lysosomes, (ii) Manders' M1 coefficient that captures mid-stage mitochondria-lysosome fusion and mitophagosome formation, and (iii) a quenching/swelling index that resolves terminal lysosomal degradation. Nutrient deprivation induced a complete, temporally ordered mitophagy program, including mtROS priming, Parkin-OPTN-associated fusion, and efficient acidification-dependent cargo degradation. In contrast, inhibition of v-ATPase with bafilomycin A1 arrested mitophagy at the fusion stage, resulting in persistent redox-active mitochondrial cargo that failed to undergo lysosomal digestion. Importantly, MitoFlare's reversible redox chemistry uniquely revealed accumulation of undegraded, oxidatively active mitochondrial remnants within non-acidified vesicles-pathological intermediates that are undetectable using irreversible ROS dyes or genetically encoded reporters. These findings demonstrate that mitophagy proceeds through discrete, redox-regulated and lysosome-dependent phases that can be quantitatively mapped in real time. By enabling synchronized measurement of oxidative activation, organelle trafficking, fusion, and degradation, the MitoFlare-LysoTracker system establishes a new benchmark for dynamic mitophagy analysis in physiologically relevant models. This platform provides a powerful foundation for mechanistic interrogation of mitochondrial quality control and for accelerating the discovery of therapeutic strategies aimed at restoring mitophagic fidelity in neurodegenerative, cardiovascular, and metabolic diseases.