FIGURE 2: Electropermeabilization by hyperpolarizing voltage steps. (A) Simultaneous monitoring of the whole-cell current and Ca²⁺ fluorescence in a cell subjected to voltage steps from -80 to -380 mV, in 20-mV increments (top plot). All steps are 25-ms long and applied at 350-ms intervals. Net pore conductance (center) is corrected for the series resistance, as described in the Methods and Fig. 3. Insets show sample Ca²⁺ transients in pseudocolor, to highlight their brightness reduction radially from the center (yellow to green). Bars are 1 µm. Fluorescence was measured in regions of interest (ROIs) where distinct Ca²⁺ transients were detected. Traces of fluorescence in all ROIs are overlapped in the bottom plot in (A), with shading applied to the area under a trace. The numbers above the traces are counts of persisting Ca²⁺ transients. (B) Same fluorescence traces plotted separately for each ROI. The red horizontal lines perpendicular to the time axis mark the voltage steps. For clarity, the time is clipped to 4.5 s. In addition to short- and long-lived Ca²⁺ transients, note in ROIs 1 and 2 subtle fluorescence fluctuations, concurrent with voltage steps and starting already at -100 mV. See text for more details.