Supplementary Figure 5: Multiple aligned pre-and postsynaptic modules represent discrete synaptic profiles within individual spines.
From: Synaptic nanomodules underlie the organization and plasticity of spine synapses
(a) A Leica TCS SP8 gated STED 3X instrument which allows for super-resolved imaging in three dimensions (~50 nm XY and ~200–250 nm in Z) was used to acquire dual-color 3D super-resolution images of endogenous PSD-95 (green) and vGlut1 (red) staining and confocal resolution images of tdTomato-filled dendritic spines (gray, white dashed outline) of DIV 21 cultured cortical neurons. Left and middle panels show orthogonal views (yellow dashed lines) of three distinct PSD-95 and vGlut1 nanomodules. Yellow, cyan and magenta arrows point to three discrete modules within the example spine. Scale bar, 1 µm. The right panel shows a 3D reconstruction of the same spine with super-resolved PSD-95 and vGlut1 nanomodules superimposed (Methods). Yellow, cyan and magenta arrows point to the 3D-rendered modules from the 2D image stacks in the left panel. Scale bar, 500 nm. Similar results were observed in at least three independent experiments. (b) Dual-color 3D super-resolved images of endogenous PSD-95 (green) and the pre-synaptic scaffolding protein Bassoon (red) staining in a confocal resolved tdTomato-filled spine (gray, white dashed outline). Orthogonal views (yellow dashed line) show two distinct, aligned PSD-95 and Bassoon modules (cyan and magenta arrows, middle panel) in the same spine. Scale bar, 1 µm. The right panel shows the 3D reconstructed image from the 2D image stacks in the left panel. Two discrete, 3D reconstructed PSD-95 and Bassoon modules are shown by cyan and magenta arrows, respectively. Scale bar, 500 nm. Similar results were observed in three independent experiments. (c-f) Positive correlation of the total synaptic area (measured as the sum of areas of all nanomodules in a spine) with the area of the spine head determined using Pearson’s correlation. All four synaptic markers (c) PSD-95 (R2 = 0.3208, p < 0.0001, F-test, n = 406 spines, CW non-gated STED at ~80 nm resolution), (d) vGlut1 (R2 = 0.2108, p < 0.0001, F-test, n = 406 spines, gated STED (AlexaFluor-594, 0.3 to 6 nsec) at ~50 nm resolution), (e) Bassoon (R2 = 0.1955, p < 0.0001, F-test, n = 217 spines, gated STED (Atto-647N, 0.2 to 6 nsec) at ~50 nm resolution) and (f) SYP-1 (R2 = 0.2860, p < 0.0001, F-test, n = 189 spines, gated STED (Atto-647N, 0.2 to 6 nsec) at ~50 nm resolution) are significantly correlated with the spine size.
