Measuring the pH of Acidic Vesicles in Live Cells with an Optimized Fluorescence Lifetime Imaging Probe
Measuring the pH of Acidic Vesicles in Live Cells with an Optimized Fluorescence Lifetime Imaging Probe
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DOI：
10.1021/acs.analchem.2c01840
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发表时间：
2022-08-01
影响因子：
7.4
通讯作者：
Urano，Yasuteru
中科院分区：
文献类型：
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作者：
Koda，Kinuko;Keller，Sascha;Urano，Yasuteru
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。大多数常规的测量pH值的方法对于量化这些囊泡内部的pH值并不令人满意。在这里，我们研究了荧光探针的分子要求，通过荧光寿命成像显微镜（FLIM）来测量活细胞中的囊内酸性pH值。开发的探针，m-DiMeNAF 488，表现出高度荧光和中等荧光形式之间的pH依赖性平衡，具有不同的和可检测的荧光寿命分别为4.36和0.58 ns。测定了m-DiMeNAF 488的pKa（τ）值为4.58，这将有利于评价酸性囊泡中的pH。我们能够通过FLIM使用m-DiMeNAF 488监测吞噬过程中吞噬体中的pH变化。该探针有望成为研究酸性pH调节的生物现象的有用工具。
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,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 pKa(τ)value ofm-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 usingm-DiMeNAF488. This probe is expected to be a useful tool for investigating acidic pH-regulated biological phenomena.