Photostable rhodamine 6G-based fluorescent probe for selective and sensitive visualization of dynamic lysosomal pH

Lysosomal pH is tightly linked to diverse cellular physiological and pathological processes, and real-time monitoring of its dynamic shifts is critical for uncovering underlying disease mechanisms. Herein, we developed a new lysosome-targeted pH fluorescent probe RGB with facile synthesis, low cytotoxicity, favorable biocompatibility and efficient cell membrane permeability. Its fluorescence emission intensity exhibits a well-defined linear correlation with environmental pH, enabling rapid, sensitive and quantitative detection of subtle pH changes at the single-lysosome level. RGB functions via a pH-triggered spirolactam ring-opening ICT fluorescence turn-on mechanism, with a measured pKa value of 4.12. Confocal fluorescence imaging results verify that RGB can evaluate oxidative stress-induced cellular damage and dynamically visualize autophagy in live tumor cells. As a robust molecular imaging reagent, RGB holds great promise for subcellular pH detection and basic mechanistic exploration of lysosome-associated disorders.