Understanding mitochondrial viscosity is crucial for comprehending cellular health and function. Therefore, accurate and sensitive measurement of mitochondrial viscosity is essential for advancing medical diagnostics and treatment strategies. In this study, a novel near-infrared (NIR) fluorescent probe SSN was developed, based on the dual mechanisms of Excited-State Intramolecular Proton Transfer (ESIPT) and Twisted Intramolecular Charge Transfer (TICT). SSN incorporates a thiophene-enhanced HBT structure and a hemicyanine moiety for effective mitochondrial targeting. It exhibits a large Stokes shift (240 nm) and high sensitivity to viscosity changes, enabling accurate detection under physiological conditions. The probe was successfully applied in HepG2 cells, zebrafish, and glucose-treated systems, demonstrating its potential for real-time viscosity monitoring in complex biological environments.
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