Characterization of single-cell electroporation by using patch-clamp and fluorescence microscopy

Electroporation of single NG108-15 cells with carbon-fiber microelectrodes was characterized by patch-clamp recordings and fluorescence microscopy. To minimize adverse capacitive charging effects, the patch-clamp pipette was sealed on the cell at a 90 degrees angle with respect to the microelectrode s where the applied potential reaches a minimum. From transmembrane current responses, we determined the electric field strengths necessary for ion-pe rmeable pore formation and investigated the kinetics of pore opening and cl osing as well as pore open times. From both patch-clamp and fluorescence mi croscopy experiments, the threshold transmembrane potentials for dielectric breakdown of NG108-15 cells, using 1-ms rectangular waveform pulses, was s imilar to 250 mV. The electroporation pulse preceded pore formation, and an alyte entry into the cells was dictated by concentration, and membrane rest ing potential driving forces. By stepwise moving a cell out of the focused field while measuring the transmembrane current response during a supramaxi mal pulse, we show that cells at a distance of similar to 30 mu m from the focused field were nest permeabilized.