Calibration of di-8-ANEPPS transients in passive fibers. (A) Di-8-ANEPPS transients (single sweeps) acquired in response to depolarizing (red traces) and hyperpolarizing voltage-clamp pulses (black traces) applied from V_H = −40 mV. Pulse amplitude was varied from −120 to 120 mV in 20-mV increments. The green trace shows the baseline noise (0 mV pulse). Fiber parameters: radius = 22 μm; length = 348 μm; capacitance: 6.6 μF/cm². (B) Steady-state amplitude of di-8-ANEPPS transients plotted as a function of the pulse amplitude. The linear regression fits to the data for hyperpolarizing and depolarizing pulses are shown as solid black and red lines, respectively. (C) Superposition of transients elicited by −120-mV (red trace) and +120-mV pulses (black trace). Both traces were normalized, and the trace in response to the −120-mV pulse was inverted. (D) Voltage dependence of the amplitude of di-8-ANEPPS transients recorded from fibers clamped at −40 mV (solid symbols, n = 4) and −20 mV (open symbols, n = 8). The slopes of the linear regressions through the solid symbols (in %/100 mV) are (mean ± SD): −13.1 ± 2.8 (black line) and −9.3 ± 2.3 (red line), for hyperpolarizing and depolarizing pulses, respectively. The slopes of the linear regressions through the open symbols are: −14.0 ± 1.7%/100mV (blue line) and −10.7 ± 1.5%/100mV (green line), for hyperpolarizing and depolarizing pulses, respectively. All data included in this figure were obtained at the end of experiments after rendering the fibers electrically passive by blocking all activatable conductances.