Sensing Mechanism and Excited-State Dynamics of a Widely Used Intracellular Fluorescent pH Probe: pHrodo

[Image: see text] The pHrodo with an “off–on” response to the changes of pH has been widely used as a fluorescent pH probe for bioimaging. The fluorescence off–on mechanism is fundamentally important for its application and further development. Herein, the sensing mechanism, especially the relevant excited-state dynamics, of pHrodo is investigated by steady-state and time-resolved spectroscopy as well as quantum chemical calculations, showing that pHrodo is best understood using the bichromophore model. Its first excited state (S(1)) is a charge transfer state between two chromophores. From S(1), pHrodo relaxes to its ground state (S(0)) via an ultrafast nonradiative process (∼0.5 ps), which causes its fluorescence to be “off”. After protonation, S(1) becomes a localized excited state, which accounts for the fluorescence being turned “on”. Our work provides photophysical insight into the sensing mechanism of pHrodo and indicates the bichromophore model might be relevant to a wide range of fluorescent probes.