Sequential dye labeling demonstrates temporally distinct endocytic zones. (A) Illustration of the dual FM dye membrane labeling assay. A micropipette first applied a focal pulse of FM 2-10 (green square, t = 0 s) and confocal images were collected for 20 s. After an additional 20 s interval, a second micropipette applied a focal pulse of FM 5-95 at time 40 s (red square) and confocal images were collected for an additional 20 s. The broken lines depict the presence of the respective FM dyes. (B-C) Time-lapse confocal images of a representative growth cone subjected to the dual FM dye labeling assay described in (A). (B) Confocal images of FM 2-10 internalization following the initial dye pulse, applied at t = 0:00, show labeled endocytic structures in the growth cone. The white arrowhead marks an endocytic zone. Time (minutes:seconds) following the FM 2-10 pulse is depicted in each frame. (B') Confocal images of the same growth cone following a second dye pulse (FM 5-95, red) show dye labeled nascent vesicles, most of which were not labeled by the prior FM 2-10 pulse. Importantly, the majority of nascent vesicles labeled by FM 5-95 (right panel, white arrows) formed in locations spatially distinct from regions where FM 2-10-positive vesicles had formed (right panel, white arrowhead). Time (min:s) following the initial FM 2-10 dye pulse is indicated in each frame. (B'') Binary images, generated from the fluorescence images in B (0:16) and B' (0:50), show distinct endocytic zones during the two FM dye pulses applied at 40-s intervals. See Additional file 11 for the time-lapse movie corresponding to B-B''. Scale bars, 5 μm.