Ehrenberg B, Montana V, Wei M D, Wuskell J P, Loew L M
Department of Physiology, University of Connecticut Health Center, Farmington 06032.
Biophys J. 1988 May;53(5):785-94. doi: 10.1016/S0006-3495(88)83158-8.
The distribution of a selection of cationic fluorescent dyes can be used to measure the membrane potential of individual cells with a microfluorometer. The essential attributes of these dyes include membrane permeability, low membrane binding, spectral properties which are insensitive to environment, and, of course, strong fluorescence. A series of dyes were screened on HeLa cells for their ability to meet these criteria and several commercially available dyes were found to be satisfactory. In addition, two new dyes were synthesized for this work by esterification of tetramethyl rhodamine. The analysis of the measured fluorescent intensities requires correction for fluorescence collected from outside the plane of focus of the cell and for nonpotentiometric binding of the dye. The measurements and analysis were performed on three different cell types for which there exists a body of literature on membrane potential; the potentials determined in this work were always within the range of literature values. The rhodamine esters are nontoxic, highly fluorescent dyes which do not form aggregates or display binding-dependent changes in fluorescence efficiency. Thus, their reversible accumulation is quantitatively related to the contrast between intracellular and extracellular fluorescence and allows membrane potentials in individual cells to be continuously monitored.
选择的一系列阳离子荧光染料的分布可用于用微荧光计测量单个细胞的膜电位。这些染料的基本特性包括膜通透性、低膜结合性、对环境不敏感的光谱特性，当然还有强荧光性。在HeLa细胞上筛选了一系列染料，以评估它们满足这些标准的能力，发现几种市售染料令人满意。此外，为这项工作通过四甲基罗丹明的酯化反应合成了两种新染料。对测量的荧光强度进行分析时，需要校正从细胞焦平面外收集的荧光以及染料的非电位结合。对三种不同类型的细胞进行了测量和分析，对于这三种细胞已有关于膜电位的大量文献；这项工作中测定的电位始终在文献值范围内。罗丹明酯是无毒、高荧光的染料，不会形成聚集体或表现出荧光效率的结合依赖性变化。因此，它们的可逆积累与细胞内和细胞外荧光之间的对比度定量相关，并允许连续监测单个细胞的膜电位。
