CONFOCAL MICROSCOPIC DETECTION OF POTENTIAL-SENSITIVE DYES USED TO REVEAL LOSS OF VOLTAGE CONTROL DURING PATCH-CLAMP EXPERIMENTS

We used a fast, fluorescent, potential-sensitive indicator (Di-8-ANEPP S) 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 relat ion between command voltage and Di-8-ANEPPS fluorescence was found in unexcitable Sf9 cells, pronounced nonlinearities were observed in card iac myocytes. Comparison of the fluorescence records and current trace s indicated that most of the observed nonlinearities could be attribut ed to voltage-escape during flow of membrane current. Voltage-escape d uring large membrane currents may lead to various experimental difficu lties during voltage-clamp experiments. The voltage recording techniqu e based on fluorescence was then used to compare the voltage-escape du ring flow of Na+ and Li+ ions via voltage-dependent (TTX sensitive) Na + channels in cardiac myocytes. In these experiments, no significant d ifferences in the degree of voltage-escape was found, suggesting that the two currents were similar in amplitude. In addition to the applica tion presented in this paper, confocal microscopic detection of transm embrane potential with fluorescent dyes may be a useful technique for experiments in preparations that are difficult to impale with microele ctrodes because of their small size.