PSD subsynaptic organization in intact neurons and isolated synaptic terminals. (A) Workflow for imaging primary cultured neurons on gold EM grids using cryo-ET. (B) Zoom-in view of the synapse in A. (C) IsoNet-corrected tomographic slice of B. Orange arrows indicate PSD density clusters. Blue region refers to a 40-nm-thick region used in PSD density analysis. (D) Density plot shows average gray values (black lines) along the 40-nm-thick blue region in the panel of C. Orange lines indicate the fitting curve of average gray values. (E) Workflow for isolating and imaging synaptoneurosomes and synaptosomes using cryo-ET. (F) A tomographic slice showing an excitatory synaptosome without IsoNet correction. (G) 3D segmentation of the synaptosome shown in F. Red arrows indicate PSD density clusters. (H) Density plot shows average gray values (black lines) along the 40-nm-thick blue region in the panel of F. (I) Cleft widths of intact synapses (n = 34), synaptoneurosomes (n = 41), and synaptosomes (n = 74) with mean ± SD of each sample shown in the figure. (J) Synaptic vesicle diameter distribution of intact synapses, synaptoneurosomes, and synaptosomes. Scale bars: (A) middle panel, 5 µm; right panel, 100 nm; (B, C, F, and G) 50 nm; (E) upper right panel, 200 nm, bottom right panel, 100 nm.