Benzothiazole-based molecular rotor as near-infrared (NIR) fluorescent probes for viscosity sensing

Intracellular viscosity is a key physicochemical parameter that reflects cellular homeostasis and pathological alterations, making it an important biomarker for disease diagnosis and monitoring. Fluorescent molecular rotors provide a powerful strategy for visualizing viscosity changes; however, many existing probes suffer from short emission wavelengths, limited tissue penetration, and suboptimal biological performance. Herein, we report a series of near-infrared (NIR) molecular rotor-based fluorescent probes (WJ-01, WJ-02, and WJ-02-IE) designed using a donor-π-acceptor architecture consisting of a dihydroxanthene-NEt₂ electron donor and a nitrile-substituted benzothiazole electron acceptor. All probes exhibited NIR emission and pronounced viscosity-dependent fluorescence enhancement governed by restricted intramolecular rotation in high-viscosity environments. Among them, WJ-02 displayed superior viscosity sensitivity and a higher fluorescence quantum yield. To improve biocompatibility, counterion engineering was employed to generate WJ-02-IE, which exhibited significantly decoupled photophysical properties from biological behavior and rapid cellular internalization. Importantly, WJ-02-IE enabled efficient imaging of intracellular viscosity changes in cancer and inflammatory cell models and preferentially localized to mitochondria. Preferential mitochondrial localization of the probes further supports their applicability for monitoring localized viscosity variations associated with metabolic and oxidative stress. Notably, successful application in a metabolic dysfunction-associated steatotic liver disease (MASLD) model highlights the capability of WJ-02-IE to probe viscosity alterations in complex pathological environments. Collectively, these results establish WJ-02-IE as an effective mitochondria-localized NIR viscosity probe for cellular and MASLD-related imaging while demonstrating counterion engineering as a useful strategy for improving the biological performance of cationic fluorescent probes.