Focal endocytic assay. (A) Illustration of the focal FM 5-95 membrane labeling assay. A focal pulse delivered from a micropipette containing FM 5-95 labels the growth cone plasma membrane. During subsequent membrane retrieval events, the lipophilic dye is trapped inside nascent vesicles. The surface membrane destains rapidly, revealing single endocytic vesicles. (B) Using a similar approach as in (A), fluorescent dextran is focally applied from a micropipette. Seconds later, a second micropipette containing buffered saline washes away the uninternalized fluorescent dextran, revealing nascent dextran-containing endocytic vesicles. (C-D) Confocal images of the same growth cone show nascent dye-containing vesicles 10 s after the focal pulse. The labeling micropipette contained both FM 5-95 (C) and Alexa488 conjugated dextran (D). The yellow arrowheads indicate endocytic vesicles exhibiting strong co-labeling. Scale bar, 5 μm.