Segmentation analysis

Free vesicles (Figure 3) were manually segmented using IMOD. Vesicles axis and volume quantifications were obtained with FIJI using the plugin LimeSeg (Machado et al., 2019) as follows: the outer contour of a vesicle was selected as the region of interest (ROI) using the “point tool” and “segmented line” tool moving in z through the tomographic slices. The lowest plane of the vesicle was annotated as a ROI with the point tool; then contours were selected with the segmented line tool every five virtual slices before finally closing the volume with the top plane of the vesicle using the point tool. LimeSeg Skeleton Segmentation tool settings were adjusted to recognize and segment the outer surface of the vesicle (D_0: 4, F_pressure: 0, Z_scale: 1, Range_in_DO_units: 1, NumberOfIntegrationStep: −1, RealXYPixelSize: 1). After the segmentation was run, the correct distribution of surfels over the outer contour of the vesicle was assessed by eye. The LimeSeg segmentation tool provides the list of vertices of the mesh. The centroid of this point cloud gives an estimate the center of the segmented vesicle. The maximum radius was then computed as the maximum distance to this center considering the 1.1 × 1.1 × 1.1–nm voxel size. Sphericity was determined according to the formula (π^1/3 × (6V)^2/3)/A. Statistical analysis was performed with Prism 7.0 (GraphPad Software).