Fig. 1.

Real-time, 3D tracking of single vesicles. (A) Immobilized Qdot (10-Hz imaging). Positional estimates had standard deviations (σ) of 8.5 nm (x), 8.5 nm (y), and 12.3 nm (z). (B) Tracking z-axial, 40-nm movements of a single Qdot in a fixed neuron. Red circle, approximate size of a vesicle. (Inset) Expanded view. (C) 3D trace of Qdot-labeled vesicle in living neuron, with x-y plane projection (red squares), overlaid on image of FM 4-64–labeled vesicles (white). Color bar, elapsed time. Stimulation (10 Hz, 120 s) started at 20 s; vesicle exocytosed at 90 s. (D) Quenching of Qdot fluorescence by TB pinpoints the moment of exocytosis. (E) Dependence of unquenched fraction (F_TB/F₀) on TB concentration. (F) 3D position and fluorescence of Qdot-labeled vesicle (exocytosis, t = 0). (G) Traces and representations of minimal, intraboutonic, and intersynaptic patterns of vesicle movement before exocytosis (G1 to G3, respectively). Average latencies to fusion were 40.7, 42.9, and 55.5 s, respectively. Gray arrows, initiation of 1200 APs stimulation. (H) Prevalence of three patterns of movement.