Mitochondrial pH (pH_mito) 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 pH_mito in live cells and tissues. This probe is designed to function by controlling the intramolecular charge transfer from 2-naphthol, having an ideal pK_a value (7.86 ± 0.05) in the cells to monitor pH_mito. 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 pH_mito in live cells and tissues. The ratiometric TPM imaging clearly reveals that subcellular distribution of the pH_mito values is heterogeneous, with the pH_mito values in the perinuclear region being higher than those at the periphery of the cells. The changes of pH_mito 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 pH_mito 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 pH_mito in biomedical research.

We report a small-molecule two-photon fluorescent probe (ANa2) for Na⁺ that shows a strong TPEF enhancement in response to Na⁺ and can be easily loaded into live cells and can real time monitor the fluctuation of [Na]_i in live cells and living tissue at more than 100 μm depth.