A membrane-potential-independent fluorescent probe with TICT-driven viscosity sensitivity for super-resolution imaging of UV-induced mitochondrial remodeling in Skin cells

Mitochondrial structural remodeling under oxidative stress is central to the pathogenesis of skin photoaging. Here, we report Mito-Py, a robust mitochondria-targeted probe with a special functional donor-π-acceptor (D-π-A) configuration. Featuring a large Stokes shift (∼130 nm) for minimal self-absorption and viscosity-sensitive emission via a twisted intramolecular charge transfer (TICT) mechanism, Mito-Py enables real-time mapping of mitochondrial viscosity fluctuations during UV-induced oxidative stress in HaCaT keratinocytes. Unlike membrane-potential-dependent dyes, Mito-Py achieves membrane-potential-independent mitochondrial retention, ensuring stable suborganellar localization even during depolarization events. Its exceptional resistance to photobleaching permits super-resolution structured illumination microscopy (SIM) to resolve UV-induced cristae fragmentation with ultrahigh spatial precision (sub-100 nm). Beyond cellular models, Mito-Py was applied to paraffin-embedded skin sections from a UV-induced murine photoaging model, where staining revealed markedly stronger fluorescence in photoaged skin compared to normal skin. These results underscore Mito-Py’s capability to detect mitochondrial alterations in complex tissue environments, providing a powerful, photostable tool for dissecting mitochondrial stress responses and exploring organelle crosstalk in photoaging and related dermatological disorders.