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A ratiometric two-photon fluorescent probe for quantitative imaging of mitochondrial pH values in live cells and tissues was reported., Mitochondrial pH (pHmito) is known to be alkaline (near 8.0) and has emerged as a potential factor for mitochondrial function and disorder. We have developed a ratiometric two-photon probe (CMP1) for quantitative analysis of pHmito in live cells and tissues. This probe is designed to function by controlling the intramolecular charge transfer from 2-naphthol, having an ideal pKa value (7.86 ± 0.05) in the cells to monitor pHmito. This transition results in a marked yellow to red emission color change in response to pH alterations from 6.0 to 9.0. CMP1 exhibits easy loading, selective and robust staining ability of mitochondria, low cytotoxicity, and bright two-photon excited fluorescence in situ, thereby allowing quantitative imaging of the pHmito in live cells and tissues. The ratiometric TPM imaging clearly reveals that subcellular distribution of the pHmito values is heterogeneous, with the pHmito values in the perinuclear region being higher than those at the periphery of the cells. The changes of pHmito values on carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment and autophagic processes were also investigated along with their morphological alterations at specific subcellular positions. We also used CMP1 to visualize the pHmito values of Parkinson's disease model astrocytes as well as living hippocampal tissues. Our results demonstrate that CMP1 will be useful as a quantitative imaging probe to study pHmito in biomedical research.