Voltage-sensitive dyes: Measurement of membrane potentials induced by DC and AC electric fields

Dye indicators of membrane potential have been available for the past 15 years and have been employed in numerous studies of cell physiology. Since the cell membrane is a likely primary site for the cascade of events resulting in a biological response to electromagnetic fields, methodologies for monitoring the membrane voltage will be critical. This laboratory has developed a series of dyes by using theoretical molecular orbital calculations to predict electrochromic structures. The spectra of electrochromic dyes are altered via a direct coupling of the molecular electronic states with an electric field. This mechanism has the advantage of providing both high temporal and high spatial resolution because the effect is instantaneous and is localized to the level of individual indicator molecules. It therefore can have significant advantages over traditional microelectrode techniques because fast changes in potential can be monitored simultaneously over many different regions of a biological preparation. We have used these dyes to monitor membrane potentials induced by both DC and AC electric fields. In a series of studies with a model membrane system, a spherical lipid bilayer, we showed that the potential develops on the membrane in good agreement with a time-dependent solution to Laplace's equation. Cell membranes can also be stained with voltage sensitive dyes. In experiments with dc fields, we are able to map the variation of the induced membrane potential along the surface of the cell by employing digital video fluorescence microscopy. We can also use the fluorescence microscope to detect membrane potential induced by ac fields by using a phase-sensitive detection scheme to extract the corresponding change in the light intensity from the fluorescent indicator. The technology can be extended to more complex biological preparations and can be used in conjunction with other optical techniques such as those which monitor intracellular ion concentrations. It may, therefore, prove highly valuable for the elucidation of biological responses to electromagnetic fields.