The speed of Lysotracker-positive cargoes is inversely correlated with their size in axons of cultured hippocampal neurons. (a) Representative time-lapse images of Lysotracker carriers at the nerve terminals of DIV14 rat hippocampal neurons. Top left: Lysotracker labeling at nerve terminals as isolated by the device. Top right: Imaris tracing trajectories of the same ROI. Trajectories of small (1#, diameter ≤0.5 µm) and large (2#, diameter >0.5 µm) carriers were magnified in the bottom panels, respectively. Rows represent the consecutive zooms of the boxed regions, from row 1 to 3. Panels in the first row, scale bar = 10 μm; second row, scale bar = 2 μm; third row, scale bar = 0.5 μm. (b) Grouped analysis of average speeds of Lysotracker-positive cargoes with small (⁠$≤$0.5 µm) and large (>0.5 µm) diameters, showing a significant difference. Data represent mean ± SEM from three independent preparations (small, n = 179; big, n = 342 tracks; ***, P < 0.001, two-tailed unpaired t test). The same datasets were also used in the pretreated group of Fig. 7 c. (c) Cumulative frequency distribution of CTB-positive cargo speed dataset used in Figs. 1 g and 7 b. (d) Cumulative frequency distribution of Lysotracker-positive cargo speed dataset used in b and Fig. 7 c.