Dye Indicators of Membrane Potential

Recently it has become possible to measure changes in the electrical poten­ tial difference across membranes of cells, organelles, and vesicles that are too small to be studied with microelectrodes. One novel technique involves the use of dye molecules as optical probes of membrane potential. Figure 1 illustrates the apparatus for observing fluorescence changes (..:1 F) and light transmission changes (..:1 J) of a preparation to which a membrane potential probe has been added. The preparation could be a single nerve or muscle fiber, many neurons in a region of brain, or a suspension of cells, organelles, or vesicles in a cuvet. As an example, Figure 2 shows the "optical action potential" of a squid giant axon stained with dye 375 (structure in Figure 3). The absorption signal of this very responsive dye is nearly superimposible on the trace of the membrane potential measured with electrodes (72). This review centers on three main classes of polymethine dyes that have provided an important source of potential-sensitive probes. These classes are the merocyanines, cyanines, and oxonols. A number of the more sensitive dyes are shown in Figure 3. Optical methods for measuring membrane potentials were reviewed earlier by Rottenberg (73), Waggoner (89), and Cohen & Salzberg (26). Membrane potential probes can also be divided into two classes based on the speed, size, and mechanism of the potential-dependent optical change. The slow dyes (or redistribution or accumulation dyes) respond to membrane potential changes in times of seconds. They are permeant and work by a mechanism involving potential-dependent redistribution of the charged dye between the medium and the inside of the cell, organelle, or vesicle. Often these dyes show absorption and fluorescence changes as large as 80-90%. Cyanine and oxonol dyes can work by this mechanism.