Measuring the pH of Acidic Vesicles in Live Cells with an Optimized Fluorescence Lifetime Imaging Probe

Acidification of intracellular vesicles, such as endosomes and lysosomes, is a key pathway for regulating the function of internal proteins. Most conventional methods of measuring pH are not satisfactory for quantifying the pH inside these vesicles. Here, we investigated the molecular requirements for a fluorescence probe to measure the in travesicular acidic pH inliving cells by means of fluorescence lifetime imaging microscopy (FLIM). The developed probe, m-DiMeNAF488, exhibits a pH-dependent equilibrium between highly fluorescent and moderately fluorescent forms, which has distinct and detectable fluorescence lifetimes of 4.36 and 0.58 ns, respectively. The pK(a(tau)) value of m-DiMeNAF488was determined to be 4.58, which would be favorable for evaluating the pH in the acidic vesicles. We were able to monitor the pH changes in phagosomes during phagocytosis by means of FLIM using m-DiMeNAF488. This probe is expected to be a useful tool for investigating acidic pH-regulated biological phenomena.

Automated summary

In this study, a new fluorescence probe, m-DiMeNAF488, was developed for measuring the acidic pH inside vesicles in living cells using fluorescence lifetime imaging microscopy (FLIM). The probe exhibits a pH-dependent equilibrium and distinct fluorescence lifetimes, allowing for the evaluation of pH changes in acidic vesicles. By using FLIM and m-DiMeNAF488, we were able to monitor the pH changes in phagosomes during phagocytosis.