Factors affecting sensitivity. A stained cell was held at 0 mV and the SEER di-8-ANEPPS signal was monitored intermittently. (A) Line-averaged R (F₂₂/F₁₁, blue) increases slightly with time during the experiment. R/R₀ during a pulse to –100 mV (red) decays in repeated pulses, so that by 25 min S_L is reduced by 50% of its initial value. (B) S_L in the cells of Fig. 4 C versus the invariant combination of F₂₂ and F₁₁ that is proportional to the concentration of dye (labeled [dye]). The correlation coefficient r² was 0.41. ID: 091412a, series 10–76.