An ultralow-concentration, red-emissive dual-responsive fluorescent probe for real-time mitochondrial viscosity/polarity imaging.

Mitochondria are central to the maintenance of cellular energy homeostasis and functional integrity, with alterations in their microenvironmental parameters being closely linked to the pathogenesis of various diseases. In particular, abnormal fluctuations in viscosity and polarity can disrupt cellular homeostasis, thereby promoting inflammation, tumorigenesis, and metabolic disorders. To overcome the limitations of existing fluorescent probes such as operational complexity and short emission wavelengths, we designed a mitochondria-targeted fluorescent probe AK based on a D-π-A molecular architecture. In this design, phenothiazine acts as the electron-donating core, a thiophene group enhances electron-donating capacity, and a positively charged indole derivative functions as the electron acceptor. The resulting probe exhibited red emission and responded dually to both viscosity and polarity. Photophysical characterization revealed that AK displayed significantly enhanced fluorescence under high-viscosity or low-polarity conditions, with a favorable linear response within specific ranges. Biological evaluation further confirmed that AK has low cytotoxicity, enables wash-free imaging, and targets mitochondria independently of membrane potential, allowing efficient imaging even at ultralow concentrations (5 nM). Moreover, AK sensitively monitored viscosity changes in both cellular and living systems, distinguished cancer cells from normal cells, and achieved precise in vivo tumor imaging. Additionally, AK enabled dynamic tracking of viscosity and polarity changes during processes such as ferroptosis and starvation, and facilitated multi-scale imaging of drug-induced liver injury across cellular, tissue and organ imaging.