Loew L M, Tuft R A, Carrington W, Fay F S
Department of Physiology, University of Connecticut Health Center, Farmington 06030.
Biophys J. 1993 Dec;65(6):2396-407. doi: 10.1016/S0006-3495(93)81318-3.
Because of its importance in the chemiosmotic theory, mitochondrial membrane potential has been the object of many investigations. Significantly, however, quantitative data on how energy transduction might be regulated or perturbed by the physiological state of the cell has only been gathered via indirect studies on isolated mitochondrial suspensions; quantitative studies on individual mitochondria in situ have not been possible because of their small size, their intrinsic motility, and the absence of appropriate analytical reagents. In this article, we combine techniques for rapid, high resolution, quantitative three-dimensional imaging microscopy and mathematical modeling to determine accurate distributions of a potentiometric fluorescent probe between the cytosol and individual mitochondria inside a living cell. Analysis of this distribution via the Nernst equation permits assignment of potentials to each of the imaged mitochondrial membranes. The mitochondrial membrane potentials are distributed over a narrow range centered at -150 mV within the neurites of differentiated neuroblastoma cells. We find that the membrane potential of a single mitochondrion is generally remarkably stable over times of 40-80 s, but significant fluctuations can occasionally be seen. The motility of individual mitochondria is not directly correlated to membrane potential, but mitochondria do become immobile after prolonged treatment with respiratory inhibitors or uncouplers. Thus, three spatial dimensions, a key physiological parameter, and their changes over time are all quantitated for objects at the resolution limit of light microscopy. The methods described may be readily extended to permit investigations of how mitochondrial function is integrated with other processes in the intact cell.
由于线粒体膜电位在化学渗透理论中的重要性，它一直是众多研究的对象。然而，值得注意的是，关于能量转导如何受到细胞生理状态调节或干扰的定量数据，仅通过对分离的线粒体悬浮液进行间接研究获得；由于单个线粒体体积小、具有内在运动性且缺乏合适的分析试剂，对其进行原位定量研究尚不可能。在本文中，我们结合快速、高分辨率、定量三维成像显微镜技术和数学建模，以确定活细胞内细胞质和单个线粒体之间电位荧光探针的准确分布。通过能斯特方程对这种分布进行分析，可将电位分配给每个成像的线粒体膜。在分化的神经母细胞瘤细胞的神经突内，线粒体膜电位分布在以 -150 mV 为中心的狭窄范围内。我们发现，单个线粒体的膜电位在 40 - 80 秒的时间内通常非常稳定，但偶尔也会出现明显波动。单个线粒体的运动性与膜电位没有直接相关性，但在用呼吸抑制剂或解偶联剂长时间处理后，线粒体确实会变得静止。因此，对于处于光学显微镜分辨率极限的物体，三个空间维度、一个关键生理参数及其随时间的变化都进行了定量。本文所述方法可很容易地扩展，以允许研究线粒体功能如何与完整细胞中的其他过程整合。
