Two-photon fluorescence visualization of lysosomal pH changes during mitophagy and cell apoptosis

We herein develop a novel two-photon fluorescent probe termed L-pH for visualization of lysosomal pH within live cells. L-pH is composed of three moieties, including naphthalimide fluorophore as a fluorescence off-on response moiety, piperazine and morpholine groups as lysosomal targeting and pH responsive sites, as well as a reactive benzyl chloride segment for further lysosomal anchoring. The experimental results demonstrate that L-pH can instantaneously respond to various pH values with high sensitivity and selectivity, and has low cytotoxicity and excellent photostability. The one-photon and two-photon fluorescence imaging data indicate L-pH can preferably accumulate into lysosome and monitor the rise of lysosomal pH changes during myriad cell stress conditions, including heat shock, cell apoptosis and mitophagy. Moreover, L-pH was applied for imaging of pH difference in abdominal tissues of mice. L-pH will be a potential tool for monitoring lysosomal pH variation during lysosome-associated physiological and pathological states.