Buck Institute for Research on Aging, 8001 Redwood Blvd, Novato, CA 94945, USA.
J Physiol. 2012 Jun 15;590(12):2845-71. doi: 10.1113/jphysiol.2012.228387. Epub 2012 Apr 10.
Mitochondrial membrane potential (ΔΨM) is a central intermediate in oxidative energy metabolism. Although ΔΨM is routinely measured qualitatively or semi-quantitatively using fluorescent probes, its quantitative assay in intact cells has been limited mostly to slow, bulk-scale radioisotope distribution methods. Here we derive and verify a biophysical model of fluorescent potentiometric probe compartmentation and dynamics using a bis-oxonol-type indicator of plasma membrane potential (ΔΨP) and the ΔΨM probe tetramethylrhodamine methyl ester (TMRM) using fluorescence imaging and voltage clamp. Using this model we introduce a purely fluorescence-based quantitative assay to measure absolute values of ΔΨM in millivolts as they vary in time in individual cells in monolayer culture. The ΔΨP-dependent distribution of the probes is modelled by Eyring rate theory. Solutions of the model are used to deconvolute ΔΨP and ΔΨM in time from the probe fluorescence intensities, taking into account their slow, ΔΨP-dependent redistribution and Nernstian behaviour. The calibration accounts for matrix:cell volume ratio, high- and low-affinity binding, activity coefficients, background fluorescence and optical dilution, allowing comparisons of potentials in cells or cell types differing in these properties. In cultured rat cortical neurons, ΔΨM is −139 mV at rest, and is regulated between −108 mV and −158 mV by concerted increases in ATP demand and Ca2+-dependent metabolic activation. Sensitivity analysis showed that the standard error of the mean in the absolute calibrated values of resting ΔΨM including all biological and systematic measurement errors introduced by the calibration parameters is less than 11 mV. Between samples treated in different ways, the typical equivalent error is ∼5 mV.
线粒体膜电位（ΔΨM）是氧化能量代谢的中心中间产物。虽然 ΔΨM 通常使用荧光探针进行定性或半定量测量，但在完整细胞中对其进行定量测定主要限于缓慢的、批量放射性同位素分布方法。在这里，我们使用荧光成像和电压钳技术，通过一种双氧杂环诺尔型质膜电位（ΔΨP）指示剂和 ΔΨM 探针四甲基罗丹明甲酯（TMRM），推导出并验证了荧光电位探针分区和动力学的生物物理模型。使用该模型，我们引入了一种纯粹基于荧光的定量测定方法，以测量在单层培养的单个细胞中随时间变化的 ΔΨM 的绝对值，单位为毫伏。探针的 ΔΨP 依赖性分布通过 Eyring 速率理论进行建模。模型的解用于从探针荧光强度中推断出 ΔΨP 和 ΔΨM 的时间变化，同时考虑到它们的缓慢、ΔΨP 依赖性重新分布和 Nernstian 行为。该校准考虑了基质：细胞体积比、高低亲和力结合、活度系数、背景荧光和光稀释，允许比较具有不同这些特性的细胞或细胞类型之间的电位。在培养的大鼠皮质神经元中，ΔΨM 在休息时为-139 mV，通过协同增加 ATP 需求和 Ca2+依赖性代谢激活，在-108 mV 和-158 mV 之间调节。敏感性分析表明，包括校准参数引入的所有生物学和系统测量误差在内的休息时 ΔΨM 的绝对校准值的平均值的标准误差小于 11 mV。在以不同方式处理的样品之间，典型的等效误差约为 5 mV。
