Fig. 1: pan-ExM reveals synapse ultrastructure in dissociated neurons.

a, NHS ester pan-stained dendrite in a nonexpanded sample showing dendritic spines. b, Axial view model of a synapse showing DPs and synaptic vesicles (SVs) in the presynaptic bouton, and the PSD, and the active zone (AZ) in the postsynaptic dendritic spine. c, Top view of a synapse showing hexagonal DPs in the presynaptic bouton and SV attachment sites. d,f–k, pan-ExM processed and NHS ester pan-stained spines including mushroom (h,j), stubby (d,f,i) and thin (g,k) shapes. e, NHS ester pan-stained synapse showing hexagonally arranged DPs. l, Transmission EM (TEM) image of a PTA-stained synapse showing prominent DPs (lime arrows) and a PSD (salmon arrow). m, pan-ExM processed and NHS ester pan-stained synapse for comparison, showing similar hallmark ultrastructural features. n, DP–PSD distances (n = 44 measurements from 4 independent experiments). o, DP–DP distances (n = 78 measurements from 6 independent experiments). p, Comparison of Bassoon–PSD and DP–PSD distances (n = 50 measurements from 3 independent samples, Welch’s two-sided t-test, P = 0.035). q, Comparison of DP–Homer1 and DP–PSD distances (n = 85 measurements from 4 independent samples, Welch’s two-sided t-test, P = 0.006). r, Comparison of PSD-95–DP and DP–PSD distances (n = 25 measurements from 2 independent samples, Welch’s two-sided t-test, P = 0.049). *P < 0.05; **P < 0.01. s, Relative spatial distributions of Bassoon, PSD-95 and Homer1 along the trans-synaptic axis. Statistical significance was assessed by Welch’s two-sided t-tests, and medians and interquartile ranges are shown with whiskers drawn down to the minimum and maximum values. t,w,z, Axial (t,w) and top (z) views of synapses pan-stained with NHS ester. u,x,aa, Bassoon immunolabeling of the same areas. v,y,bb, Respective overlays. cc–kk,ll–qq,rr–ww,xx–ccc, Same as t–bb in samples labeled for Homer1, labeled for PSD-95, double-labeled for Homer1 and Bassoon, and labeled for Synaptophysin (SYN), respectively. The inset in ccc shows SYN puncta, representing synaptic vesicles, intercalated between neighboring DPs. ddd, NHS ester image of a mitochondrion in a hippocampal rat neuron. mtDNA, mitochondrial DNA. eee, SYTOX Green (SYX) staining of the same area. fff, Overlay. ggg, NHS ester image of a nuclear pore complex (NPC). hhh, Overlay of ggg with a SYTOX Green image of the same area. iii, NHS ester image of basal bodies in a mouse neuron. The inset shows the familiar centriolar cartwheel structure. jjj, NHS ester image of a cilium in a mouse neuron. Lime and salmon arrows point to the basal body and ciliary tip, respectively. Medians and interquartile ranges are shown with whiskers drawn down to the minimum and maximum values. Panels showing pan staining or TEM (l) are displayed with a white-to-black color table; panels showing immunolabeling or SYTOX Green staining are displayed with a black-to-white color table. Gamma corrections: d,e, γ = 0.8; h,j,k,m,cc,ll,oo,rr,uu,xx,aaa,iii,jjj, γ = 0.7; i, γ = 0.6. jjj is a z-projection (intensity average) of five images. All scale bars are corrected for the expansion factor. Scale bars: 800 nm (a), 200 nm (d–m,t–ccc, jjj), 300 nm (ddd–fff), 50 nm (ggg,hhh), 100 nm (iii). Expansion factor: 16 ± 2.