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

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Measuring the pH of Acidic Vesicles in Live Cells with an Optimized Fluorescence Lifetime Imaging Probe
Analytical Chemistry ( IF 6.7 ) Pub Date : 2022-08-01 , DOI: 10.1021/acs.analchem.2c01840
Kinuko Koda , Sascha Keller , Ryosuke Kojima ₁ , Mako Kamiya ₂ , Yasuteru Urano ₃

Affiliation

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 pK_a(τ) value of m-DiMeNAF488 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 m-DiMeNAF488. This probe is expected to be a useful tool for investigating acidic pH-regulated biological phenomena.

中文翻译：

使用优化的荧光寿命成像探针测量活细胞中酸性囊泡的 pH 值

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。大多数测量 pH 值的常规方法不能令人满意地量化这些囊泡内的 pH 值。在这里，我们研究了通过荧光寿命成像显微镜 (FLIM) 测量活细胞中囊泡内酸性 pH 值的荧光探针的分子要求。开发的探针m -DiMeNAF488在高荧光和中等荧光形式之间表现出 pH 依赖性平衡，其荧光寿命分别为 4.36 和 0.58 ns，可检测到不同的荧光寿命。m -DiMeNAF488的 p K _a(τ)值确定为4.58，这将有利于评估酸性囊泡中的pH值。我们能够通过 FLIM 使用m -DiMeNAF488监测吞噬过程中吞噬体的 pH 变化。该探针有望成为研究酸性 pH 调节生物现象的有用工具。

更新日期：2022-08-01

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