Fig. 4. TIRFM image of a growth cone (A) showing the distribution of NCS-1-EYFP on plasma-membrane-apposed puncta before electrical stimulation. Bar, 2 µm. (B) Images showing the changes in the appearance of a vesicle that underwent stimulation-dependent exocytosis. 33 milliseconds after application of the stimulus, the vesicle fluorescence was widened relative to its initial fluorescence, and 200 milliseconds after application of the stimulus, the vesicle essentially was invisible as its associated fluorescence had spread into the background. Bar, 1 µm. Individual (C) and averaged (D) curves showing fluorophore movement toward the plasma membrane and spreading of fluorescence after electrical stimulation. Data obtained from circles and annuli are represented by closed and open circles, respectively. Fluorescence is expressed in pixel units (P.U.). Bars, 200 milliseconds and 50 milliseconds.