A two-photon ratiometric fluorescent probe enables spatial coordinates determination of intracellular pH

We reported a two-photon ratiometric fluorescent probe for detecting intracellular pH. When excited with 800   nm laser, an optimal output of laser as the routine equipment of two-photon fluorescence microscopy , the two-photon excited fluorescence of this probe showed distinct emission peak shift as large as 109   nm upon the change of pH values in vitro. Very importantly, the experiment results show that this probe has large two-photon absorption cross-section at pH 4.5 at 800   nm of 354   g, which ranks it as one of the best two-photon ratiometric fluorescent pH probes, and its working pH value is between 4.0 and 8.0 which could fit the intracellular pH range. Moreover, utilizing this probe, the two-photon ratiometric fluorescent images in living cells have been obtained, and the spatial coordinates of intracellular pH can be mapped. At the same time, the probe also exhibited selectivity, photostability and membrane permeability. And the photophysical properties of this probe in various solvents indicated that these photophysical properties variations are due to an intramolecular charge transfer process. At last, the imaging depth of the probe in liver biopsy slices was investigated. The experimental results demonstrated the maximum imaging depth can arrive 66   µm in living rat liver tissues.