Dual-Modality Imaging Unveil Inner Mitochondrial Membrane Viscosity and Respiratory Dynamics in Mitophagy

Mitophagy is a vital lysosome-dependent process that maintains mitochondrial integrity and cellular homeostasis, where respiration and inner mitochondrial membrane (IMM) viscosity play key roles. Despite its critical importance, achieving a high-resolution and dynamic visualization of respiration and IMM viscosity during mitophagy remains a significant challenge. In this study, we designed two innovative fluorescent probes: SiR-C8, a viscosity-sensitive rotor-type probe based on silicon-rhodamine, specifically targeting the IMM, and OR-ATP, a rhodamine-derived probe utilizing an intramolecular spirolactam structure to respond to mitochondrial ATP levels. Leveraging fluorescence intensity and lifetime dual-modality imaging, we successfully enabled the high-resolution, real-time monitoring of lysosome-dependent mitophagy. Remarkably, our results unveiled a progressive increase in IMM viscosity alongside a significant attenuation in mitochondrial respiration during mitophagy induced by starvation, carbonyl cyanide, <i>m</i>-chlorophenyl hydrazone (CCCP), and Oligomycin. Significantly, utilizing structured illumination microscopy super-resolution imaging, we have uncovered a novel mitochondrial quality control mechanism by which lysosomes selectively engulf locally damaged mitochondrial regions. This discovery provides novel insights into the intricate processes governing mitophagy and introduces an innovative platform for studying mitochondrial dynamics, dysfunction, and their implications for cellular homeostasis and pathology.