A Dual-Organelle Fluorescent Probe for Visualizing Intracellular Microviscosity Dynamics.

Intracellular microviscosity is a key physicochemical parameter that influences molecular diffusion, signal transduction, and organelle function. However, fluorescence probes capable of selectively visualizing viscosity dynamics with high sensitivity and biological compatibility remain limited. Here, we report a dihydroxanthene-based viscosity-responsive fluorescent probe, ZKW, constructed with a D-π-A push-pull architecture and an o-bromophenyl molecular rotor. ZKW displays a pronounced viscosity-dependent fluorescence enhancement at 580 nm upon 550 nm excitation, originating from restricted intramolecular rotation and suppressed TICT processes. The probe exhibits good selectivity toward viscosity with negligible interference from polarity, pH, reactive species, metal ions, amino acids, or proteins, along with acceptable photostability and low cytotoxicity. Colocalization studies revealed preferential mitochondrial accumulation with partial lysosomal distribution, enabling simultaneous visualization of viscosity variations in these organelles. Using multiple cellular stress models-including LPS stimulation, oleic-acid treatment, nystatin exposure, and erastin-induced ferroptosis-ZKW effectively reported viscosity elevations at the subcellular level. These results establish ZKW as a robust and versatile tool for probing microviscosity heterogeneity in living cells and for facilitating studies of viscosity-associated physiological and pathological processes.