FIGURE 1: Electropore detection by imaging Ca²⁺ transients. (A) A schematic of the setup that enables TIRF imaging of electroporated cell membrane. A cell is placed on a transparent indium tin oxide (ITO) electrode on a glass coverslip and loaded with the Ca²⁺ fluorophore CAL-520 via a patch clamp pipette. Voltage steps applied between the pipette and the ITO create membrane lesions that admit Ca²⁺ into the cell producing fluorescence transients (pictured by small green spheres). (B and C) TIRF images of Ca²⁺ transients in two cells subjected to hyperpolarizing voltage steps. Each step was from zero to the indicated voltage (mV) and lasted 25 ms. The leftmost images show a subthreshold stimulation with no visible fluorescence spots. In the next images, arrows highlight the transients that were not necessarily re-evoked by larger voltage steps. The arrows maintain the same position across all the images. Bar: 10 µm. In panel B, the brightness of the three images on the left was enhanced for visual clarity.