Behavior of individual vesicles visualized using TIRFM. (A) Sequence of images (40 ms/frame) showing a single vesicle loaded with 10-kD Alexa Fluor 488 as it approached the membrane and then fused. (B) Image sequence showing a vesicle that approached the membrane and then departed without fusion. Bars, 0.5 µm. (C) Changes in fluorescent brightness as a function of time plotted for a 7 × 7–pixel region of interest centered on the vesicle in A. Brightness increased as the vesicle approached the membrane and then declined abruptly after dye was released by fusion. Graph plots the fluorescence changes in arbitrary units (a.u.) measured at −340 to 80 ms (arrows). (D) Changes in fluorescent brightness as a function of time for the vesicle in B. As the vesicle retreated from the membrane, its brightness declined at roughly the same rate as it increased during membrane approach. Graph plots the fluorescence changes measured at −200 to 240 ms (arrows). (E) Fluorescence changes as a function of time from a vesicle that was stably associated with the membrane and then fused, causing its fluorescence to decline abruptly. (F) Fluorescence changes from a vesicle that was stably associated with the membrane but then departed without fusion, showing a gradual decline in fluorescence. These examples are all from rods loaded with 10-kD Alexa Fluor 488.