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(o) angle with respect to the microelectrodes where the applied potential reaches a minimum. From transmembrane current responses, we determined the electric field strengths necessary for ion-permeable pore formation and investigated the kinetics of pore opening and closing as well as pore open times. From both patch-clamp and fluorescence microscopy experiments, the threshold transmembrane potentials for dielectric breakdown of NG108-15 cells, using 1-ms rectangular waveform pulses, was approximately 250 mV. The electroporation pulse preceded pore formation, and analyte entry into the cells was dictated by concentration, and membrane resting potential driving forces. By stepwise moving a cell out of the focused field while measuring the transmembrane current response during a supramaximal pulse, we show that cells at a distance of approximately 30 microm from the focused field were not permeabilized.
Ryttsén
F
F
Department of Chemistry, Göteborg University, Göteborg SE-412 96, Sweden.
Farre
C
C
Brennan
C
C
Weber
S G
SG
Nolkrantz
K
K
Jardemark
K
K
Chiu
D T
DT
Orwar
O
O
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002460:Cell Line; D002463:Cell Membrane Permeability; D004563:Electrochemistry; D018274:Electroporation / Q000295:instrumentation / Q000379:methods*; D007700:Kinetics; D008564:Membrane Potentials; D008839:Microelectrodes; D008856:Microscopy, Fluorescence; D018408:Patch-Clamp Techniques