Monitoring pH in lysosomes of living cells using a pH-sensitive probe based on spirocyclization strategy

The pH-sensitive fluorescent probe CBTOH, utilizing a spirocyclization mechanism, has been developed for precise pH monitoring and imaging in biological systems. Under acidic conditions (pH 2-3.5), CBTOH undergoes ring-opening, emitting red fluorescence at 611 nm with a 119-fold increase in intensity compared to neutral and alkaline conditions, due to enhanced intramolecular charge transfer upon spirolactam ring cleavage. The probe exhibits excellent photostability, reversibility, and selectivity for pH changes, with minimal interference from common biological ions and amino acids. Confocal fluorescence imaging in HeLa cells and zebrafish models confirms its lysosome-specific targeting and sensitivity to pH fluctuations, especially in acidic environment. CBTOH effectively monitors drug-induced endogenous pH changes, such as those induced by rapamycin, thereby offering a novel methodological approach for investigating drug-induced physiological alterations associated with lysosomal activity.