The steady SEER signal of di-8-ANEPPS under voltage clamp. (A) F₂₂(x,y), indicating the scanned line (broken line) in a cell under voltage clamp held at 0 mV. (B) SEER ratio image R(x,t), normalized to its resting value R₀(x), when the cell in A was subjected to a biphasic pulse consisting of hyperpolarization to −160 mV for 400 ms followed by depolarization to +160 mV, as illustrated in C. (C) Line averages (R(t)/R₀) in the same cell, subjected to the biphasic pulses levels coded by color. (D) Average level of R/R₀ during the pulse in the cell of A–C (black circles) and six others. The average slope was −0.15 (100 mV)⁻¹, with a standard deviation of 0.017. ID of cells 1–7: 091412a, -b, -c, -d, 111011a, 091712b, and 091712a.