A Sensitive Fluorescent Probe for Tracking Viscosity Changes in the Mitochondria Under Physiological and Pathological Conditions

ABSTRACT Mitochondrial viscosity is a key microenvironmental parameter regulating its function, and its dysregulation is associated with various diseases. However, achieving specific and dynamic monitoring of mitochondrial viscosity remains challenging. To address this, we developed a novel near‐infrared fluorescent probe, FTZ‐BTZ , based on a twisted intramolecular charge transfer mechanism. The probe demonstrates high sensitivity ( R 2 = 0.9906), excellent selectivity, good stability, and low cytotoxicity in viscosity detection. Colocalization imaging confirmed its precise mitochondrial targeting capability, with a Pearson's coefficient of 0.9052. Using this probe, we successfully achieved real‐time, in situ visualization of dynamic changes in mitochondrial viscosity within living cells. These changes were induced by various pharmacological stimuli, including lipopolysaccharide, dexamethasone, nystatin, and monensin. Specifically, we established the concentration‐dependent response of mitochondrial viscosity to monensin. Further in vivo experiments showed that FTZ‐BTZ can effectively distinguish viscosity gradients induced by different drug stimulations in mouse models. The FTZ‐BTZ probe developed in this work provides a high‐performance molecular tool for real‐time investigation of mitochondrial viscosity‐related physiological and pathological processes at the subcellular level.