Internalized VAMP2–pHluorin/Atto647N nanobodies exhibit lower mobility compared with those transiting on the plasma membrane. Hippocampal neurons were transfected with VAMP2–pHluorin and subjected to sdTIM or uPAINT, and the mobility of VAMP2–pHluorin–bound Atto647N nanobody after internalization was compared with VAMP2–pHluorin/Atto647N nanobody mobility on the plasma membrane (PM). The graphs show MSD (A; µm²), AUC (B; in arbitrary units [a.u.]), and frequency distribution (C) of the mean Log₁₀D. The threshold for the mobile (Log₁₀D ≤ −1.45) and immobile fraction (Log₁₀D > −1.45) is indicated with a dashed line. The inset in C shows the mobile-to-immobile (M/IMM) ratios of VAMP2–pHluorin/Atto647N nanobodies internalized in SVs and transiting on the plasma membrane. n = 12 hippocampal neurons in individual cultures in sdTIM (97,400 trajectories) and uPAINT (52,800 trajectories). Statistical analyses of independent experiments were performed using the Student’s t test (C, inset) and Mann–Whitney U test (B). Error bars are ±SEM. See also Fig. S2 A.