A pH-Resilient fluorogenic probe for stable high-resolution imaging of lysosomal dynamics

Rhodamines are widely utilized for fluorescence switching, biosensing, and super-resolution imaging due to their dynamic equilibrium between a non-fluorescent "closed" spiro-ring form and a fluorescent "open" xanthylium form. This equilibrium has inspired extensive efforts to chemically modulate their spiro-ring behavior. In this study, we uncover a novel mechanism in aniline-rhodamine ( PR ), where the interplay of NH protonation, deprotonation, and hydrogen bonding collaboratively governs spirolactone ring opening. Notably, this interplay stabilizes the ring equilibrium in aqueous solutions within the pH range of 4–10, minimizing sensitivity to pH fluctuations. Leveraging this mechanism, we designed an aniline-rhodamine derivative, EPR , which maintains stable fluorescence regardless of lysosomal pH variations. This stability enables consistent, high-resolution imaging of lysosomal dynamics under both physiological and pathological conditions, offering deep insights into lysosomal functions within cellular regulatory networks. The NH groups in PR establish a new regulatory axis for rhodamine ring switching, providing a promising foundation for developing next-generation fluorescent probes with enhanced stability and functionality.