Mitochondria-immobilized fluorescent probes for viscosity and cellular imaging

A mitochondria-immobilized fluorescent probe Mito-CDM was designed and synthesized for viscosity sensing via the twisted intramolecular charge transfer (TICT) mechanism. The N , N -diethylaminophenyl group is the viscosity-sensitive unit in the prepared Mito-CDM molecular structure, while the pyridinium cation is the mitochondria-targeting group that is responsible for monitoring mitochondrial viscosity. As the viscosity increased from 0.55 cp (0% glycerol) to 950 cp (100% glycerol), the fluorescence intensity of Mito-CDM at 586 nm enhanced gradually and there is a good linear relationship between fluorescence intensity and viscosity ( n  = 11, R 2  = 0.9957). In high-viscosity media, Mito-CDM exhibits a 166-fold fluorescence enhancement at 586 nm. It is crucial to note that the benzyl chloride group has the ability to react with highly reactive thiols on mitochondrial proteins, which enables Mito-CDM to be immobilized on mitochondria for extended periods. Additionally, Mito-CDM was successfully used to monitor mitochondrial viscosity changes during nystatin and lipopolysaccharide-induced inflammation in HeLa cells.