Average vesicle behavior visualized by TIRFM. (A) Sequence of images (40 ms/frame) showing release events averaged from 16 vesicles. (B) Sequence of images (40 ms/frame) showing nonrelease retreat events averaged from 20 vesicles. Bars, 0.5 µm. (C) Changes in fluorescent brightness as a function of time measured within a 7 × 7–pixel region of interest centered on the average vesicle images shown in A and B. Brightness declined much more rapidly after fusion than when vesicles retreated from the membrane without fusion. (D) Histogram plotting the number of release events before and after application of a depolarizing step (−70 to −10 mV, 100 ms applied at 1 s) to voltage-clamped rods (52 events in 7 rods). (E) Histogram plotting the number of nonrelease retreat events before and after application of a depolarizing step (−70 to −10 mV, 100 ms) to voltage-clamped rods (80 events in 7 rods). For experiments in this figure, rods were loaded with 3-kD Alexa Fluor 488.