Wash-free and ultra-low concentration monitor lysosomal viscosity in apoptosis with a noteworthy fluorescent probe.

The visualization and subsequent monitoring of apoptosis holds paramount significance in the domains of physiology, pathology, and pharmacology. However, traditional probes require high staining concentrations and multiple washing steps, which would alter the specimen's micro-environment, potentially inducing harm to specimen. To overcome these challenging issues, we have rationally designed and prepared a pH-inert lysosomal probe (named IVTI) to wash-free visualize apoptosis with ultra-low concentration to alleviate the disturbance of probe concentration, washing procedure and pH variations. Compared with general lysosomal probes, IVTI showed a significant fluorescence boost in reflex to elevated viscosity, while its fluorescence intensity remained mostly still when altering pH values, which could achieve more accurate visualization of lysosomes. Moreover, the probe can detect minute viscosity fluctuations in lysosomes under extra-low concentration, greatly eliminating the effect of probe concentration and washing steps to live bio-samples. Furthermore, compared to LTR (Lyso-Tracker Red, a commercial lysosome probe), IVTI offered exceptional imaging capabilities, and the fluorescence images of IVTI was still clear when lysosomal pH increased, which attributed to the pH-inert properties of IVTI. In view of the excellent imaging abilities, the pH-inert probe was applied to in-situ and real time visualize viscosity changes of live cells under extra-low concentration without washing procedure, and the increase of lysosomal viscosity during apoptosis was also monitored by the probe, thereby minimizing the disturbance of probe concentration, washing procedure and pH variations during apoptosis. The probe possesses tremendous potential in the visualization of dynamic changes related to lysosomes in various physiological processes.