Restimulation increases the SV mobility in presynapses to that of VAMP2–pHluorin mobility on the plasma membrane. The mean MSD (A; µm²), AUC (B; arbitrary units [a.u.]), and mean frequency distribution of Log₁₀D and the mobile-to-immobile (M/IMM) fraction (C) after restimulation in axonal segments (Ax) and presynapses (Ps). n = 113 presynapses and 148 axonal segments from 23 hippocampal neurons in individual cultures (7,800 and 21,500 trajectories, respectively). (D–F) Comparison of VAMP2–pHluorin/Atto647 nanobody mobility after restimulation to that detected on the plasma membrane (measured with uPAINT) in presynapses. Imaging was restricted to 120 s in both conditions. n_uPAINT = 50 presynapses from 12 hippocampal neurons (6,400 trajectories) and n_{restimulation} = 113 presynapses from 23 hippocampal neurons (7,800 trajectories) in individual cultures. Statistical analyses of independent experiments were performed using the Student’s t test (C and F, insets) or Mann–Whitney U test (B and E). Error bars are ±SEM. See also Fig. S2 F.