The size of axonal cargoes correlates with the diameter of the axon. (a) Bright-field image of DIV14 rat hippocampal neurons cultured in a microfluidic device with the region selected for EM outlined. Scale bar = 250 µm. (b) Representative electron micrographs showing the axonal diameter measurements with cargo (red) or without cargo (blue) and the associated cargo size (white). Scale bar = 0.5 µm. (c) Electron micrographs of axon bundles from hippocampal neurons cultured in microfluidic devices. i–iv: Axon diameters with and without cargo are marked with red and blue arrows, respectively. i: Endosome (black arrow). i–iii: Mitochondria (white arrowheads) and autophagosome (black arrowheads). Iv: Inner diameters of cargoes are marked with white arrows. Scale bars = 500 nm. (d) Quantification of axon diameters with cargo and without cargo. Data represent mean ± SEM, n = 182 (+ Cargo) and 182 (− Cargo) measurements from two independent preparations (***, P < 0.001, two-tailed paired t test). (e) Grouped quantification of axonal diameter as a function of binned vesicle size. Data represent mean ± SEM; for the + Cargo group from left to right: n = 104, 23, 41, and 14 measurements; for the − Cargo group from left to right: n = 104, 23, 41, and 22 measurements. Data are from two independent preparations (*, P < 0.05; ***, P < 0.001, two-tailed unpaired t test). (f) Cross-correlation analysis of cargo size and axonal diameter. Linear regressions were performed with the 182 paired measurements of axonal and cargo diameters of the + Cargo group. Data are from two independent preparations.