Comparison of squid synaptic vesicle (SV) size by altering electron microscopy sample preparation technique in synaptosomes and isolated vesicles. (A) Electron micrograph of (left to right) fixed, embedded, thin-sectioned synaptosome; slam-frozen, freeze substituted synaptosome; negative stained isolated SVs and slam-frozen, freeze substituted isolated synaptic vesicles. (B) Cumulative distribution of synaptic vesicle diameters. Variations in mean synaptic vesicle diameter due to different sample preparation regimens (mean ± SEM, n= 125-175, Student's t-test; “*” = p< 0.03, “***” = p<0.001, n.s. = not significant).