Real-Time Monitoring Mitochondrial Viscosity during Mitophagy Using a Mitochondria-Immobilized Near-Infrared Aggregation-Induced Emission Probe

Mitophagy plays a crucial role in maintaining intracellular homeostasis through the removal of dysfunctional mitochondria and recycling their constituents in a lysosome-degradative pathway, which leads to microenvironmental changes within mitochondria, such as the pH, viscosity, and polarity. However, most of the mitochondrial fluorescence viscosity probes only rely on electrostatic attraction and readily leak out from the mitochondria during mitophagy with a decreased membrane potential, thus easily leading to an inaccurate detection of viscosity changes. In this work, we report a mitochondria-immobilized NIR-emissive aggregation-induced emission (AIE) probe CS-Py-BC, which allows for an off-on fluorescence response to viscosity, thus enabling the real-time monitoring viscosity variation during mitophagy. This system consists of a cyanostilbene skeleton as the AIE active core and viscosity-sensitive unit, a pyridinium cation for the mitochondria-targeting group, and a benzyl chloride subunit that induces mitochondrial immobilization. As the viscosity increased from 0.903 cP (0% glycerol) to 965 cP (99% glycerol), CS-Py-BC exhibited an about 92-fold increase in fluorescence intensity at 650 nm, which might be attributed to the restriction of rotation and inhibition of twisted intramolecular charge transfer in a high viscosity system. We also revealed that CS-Py-BC could be well immobilized onto mitochondria, regardless of the mitochondrial membrane potential fluctuation. Most importantly, using CS-Py-BC, we have successfully visualized the increased mitochondrial viscosity during starvation or rapamycin-induced mitophagy in real time. All these features render CS-Py-BC a promising candidate to investigate mitophagy-associated dynamic physiological and pathological processes.