Confocal microscopic detection of potential-sensitive dyes used to reveal loss of voltage control during patch-clamp...

We used a fast, fluorescent, potential-sensitive indicator (Di-8-ANEPPS) in combination with laser-scanning confocal microscopy in the line-scan mode (temporal resolution 500 Hz) to independently determine the transmembrane potential in voltage-clamped cells. While a linear relation between command voltage and Di-8-ANEPPS fluorescence was found in unexcitable Sf9 cells, pronounced nonlinearities were observed in cardiac myocytes. Comparison of the fluorescence records and current traces indicated that most of the observed nonlinearities could be attributed to voltage-escape during flow of membrane current. Voltage-escape during large membrane currents may lead to various experimental difficulties during voltage-clamp experiments. The voltage recording technique based on fluorescence was then used to compare the voltage-escape during flow of Na+ and Li+ ions via voltage-dependent (TTX sensitive) Na+ channels in cardiac myocytes. In these experiments, no significant differences in the degree of voltage-escape was found, suggesting that the two currents were similar in amplitude. In addition to the application presented in this paper, confocal microscopic detection of transmembrane potential with fluorescent dyes may be a useful technique for experiments in preparations that are difficult to impale with microelectrodes because of their small size.