Iodide quenching experiments highlight differences in solvent accessibility for each fluorophore. (A) Recordings of ΔF under control conditions (left traces) and following incubation in a solution for 30 s supplemented with 50 mM KI (see Materials and methods) for representative ALEXA-488–labeled oocyte. Upper traces: absolute fluorescence relative to the dark state; lower traces: an expanded view of ΔF in response to a voltage step to +60 and −160 mV from 0 mV holding voltage. Arrows indicate ∆F_fast component that is reduced after I⁻ exposure (see Fig. S3). ΔF shown as percentage of background fluorescence. (B) ∆F_total plotted as a function of membrane potential pooled from (n = 9) cells and fit with the Boltzmann equation. Data were normalized to the predicted $∆Ftotalmax$ from the fit to the control data set for each cell and pooled. The fit parameters are given in Table 3. (C) Recordings of fluorescence under control conditions (left traces) and following incubation in a solution for 30 s supplemented with 50 mM KI (see Materials and methods) for a representative MTS-TAMRA–labeled oocyte. Upper traces: absolute fluorescence relative to the dark state; lower traces: an expanded view of ΔF in response to a voltage steps to +60 and −160 mV from 0 mV holding voltage. ΔF shown as percentage of background fluorescence. (D) ∆F_total plotted as a function of membrane potential pooled from (n = 11) cells and fit with the Boltzmann equation. Data were normalized to the predicted $∆Ftotalmax$ from the fit to the control data set for each cell and pooled. The fit parameters are given in Table 3.