A ratiometric fluorescent probe with dual red/near-infrared emissions for monitoring lysosomal pH fluctuations

Lysosomal pH is a crucial intracellular parameter for evaluating cellular metabolism, as even minor fluctuations are closely associated with various physiological disorders. Ratiometric fluorescent probes have been developed for monitoring lysosomal pH changes through molecular imaging. However, most of the reported probes suffer from limitations such as inadequate intracellular calibration and short excitation and emission wavelengths, which hinder their accurate pH quantification and restrict their applicability <i>in vivo</i>. Here we report a ratiometric fluorescent probe HeCypH, which was synthesized <i>via</i> a simple approach, to track pH fluctuations. The probe design relies on a hemicyanine scaffold fused with an intramolecular oxazinane ring, which undergoes a pH-responsive ring-opening process. Under acidic conditions, the open-ring form HeCypH-O exhibits two red/near-infrared emission peaks at 615 nm and 722 nm. Confocal fluorescence imaging in living cells revealed that HeCypH selectively localizes in lysosomes and enables ratiometric measurement of intracellular pH with high precision. Moreover, the excellent pH sensitivity, reversibility, and red-shifted emissions of HeCypH make it well suited for real-time monitoring of pH fluctuations and deep-tissue bioimaging in a mouse model.