Figure 3
From: Volumetric, Nanoscale Optical Imaging of Mouse and Human Kidney via Expansion Microscopy
Quantification of GBM thickness and FP width. (a) Volume perspective rendering of expanded mouse kidney immunostained for podocin (green), agrin (red), podocalyxin (blue), and stained for DNA with Hoechst (white), highlighting podocyte foot process (FP) boundaries, glomerular basement membrane (GBM), endothelial cells, the apical side of FPs, and nuclei, respectively. See Supplementary Movie 1 for an animation of this volume. (b) A single confocal image plane from data shown in (a). (c) Zoomed-in view of podocin channel from boxed region in (b) showing two methods for FP width (FPW) analysis. (d) Line profile (Int., signal intensity) of line in (c) (arb., arbitrary units) including a fit of the profile (black dots) with several Gaussians (green line) for determining positions of FPs. (e) Histogram of FPW determined by measuring peak-to-peak distances in (d) (data from 2 kidney samples, 7 glomeruli, and 61 profiles). (f) Histogram of average FPWs (data from 2 kidney samples, 7 glomeruli, and 39 profiles) determined by dividing the area within a flat region of the glomerulus (yellow dashed line) by the length of the podocin signal within that area (red lines in c). (g) Zoomed-in view of boxed region in (b) where the basement membrane and FPs are approximately perpendicular to the image plane (nuclear channel omitted for clarity). (h) Cross-sectional profile of the (green) podocin channel in the boxed region of (g). (i) Zoomed-in view of the podocalyxin (blue) and agrin (red) channels from the boxed region in (b). (j) Cross-sectional profile (points) of the boxed region in (i) together with Gaussian fits (lines). (k) Histogram of GBM thicknesses measured as the full width at half maximum of multiple agrin profiles (data from 3 kidney samples, 4 glomeruli, and 154 profiles). All distances and scale bars are in pre-expansion units. Scale bars, 8 µm (b), 2 µm (c), 1 µm (g), and 500 nm (i).
