PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.
Department of Life Science and Technology, Tokyo Institute of Technology, 4259, Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.
Anal Chem. 2022 Aug 16;94(32):11264-11271. doi: 10.1021/acs.analchem.2c01840. Epub 2022 Aug 1.
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 intravesicular acidic pH in living cells by means of fluorescence lifetime imaging microscopy (FLIM). The developed probe, , 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 p value of was 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 . This probe is expected to be a useful tool for investigating acidic pH-regulated biological phenomena.
细胞内囊泡（如内体和溶酶体）的酸化是调节内部蛋白质功能的关键途径。大多数传统的测量 pH 值的方法都不能令人满意地定量测量这些囊泡内的 pH 值。在这里，我们通过荧光寿命成像显微镜（FLIM）研究了一种荧光探针测量活细胞内囊泡酸性 pH 值的分子要求。所开发的探针 ，在高度荧光和中度荧光形式之间表现出 pH 依赖性平衡，分别具有明显且可检测的荧光寿命 4.36 和 0.58 ns。 的 p 值确定为 4.58，这有利于评估酸性囊泡中的 pH 值。我们能够通过使用 进行 FLIM 监测吞噬作用过程中吞噬体的 pH 值变化。该探针有望成为研究酸性 pH 值调节的生物学现象的有用工具。
