Novel PSD-95 nanodomains as building blocks of postsynaptic membrane regions. (A and B) Live-cell imaging of PF11-GFP by STED microscopy (green), but not by conventional confocal microscopy (red pseudocolor), efficiently detects multiple subspine clusters (1 to 4 clusters/spine) in neurons. Bars: (A) 1 µm; (B) 500 nm. (C and D) Size of subsynaptic nanodomains. Subsynaptic clusters of PF11-GFP and PSD-95-GFP visualized by live STED imaging were measured across the longest axis. Approximately 200 clusters were randomly selected from 10 live-imaging experiments. FWHM, full width at half maximum. ***, P < 0.001 by Student’s t test. (E) The size of postsynaptic membrane regions (determined by confocal microscopy, red in B, according to diameter at the longest axis) correlates well with the number of PF11-labeled subsynaptic nanodomains (determined by STED, green in B). 130 postsynapses from 5 live neurons were analyzed. (F) Dual-color STED (2C-STED) analysis of fixed neurons. Postsynaptic hPF11 labeling (red pseudocolor) shows nanodomain structures (arrowheads), overlapping with total PSD-95 labeling (green pseudocolor, top panels) and apposed to presynaptic vGlut1 labeling (green pseudocolor, bottom). The same fields were sequentially imaged in the confocal mode. Bars, 500 nm. (G) The size of PF11-labeled postsynaptic regions was measured by STED microscopy, as described in Fig. S2 B for a representative image from F, and the frequency histogram is shown. Peaks by the Gaussian approximation centered at 225, 420, and 625 nm are shown in red dashed lines. In total, 450 clusters from 6 neurons (three independent experiments) were analyzed. Representative regions with one, two, and three nanodomains are shown (insets). Bars, 200 nm. (H) Individual nanodomains (red pseudocolor) in a spine are associated with AMPARs (green pseudocolor). sGluA1, surface-expressed GluA1. Arrows, nanodomains labeled by PF11-Venus. Bar, 500 nm.