Fig. 3: 3D imaging and virtual flattening of neuronal plexuses in the mouse gut.
From: enGLOW 3D microscopy of the enteric nervous system in cleared human and mouse gut
a Two-channel mesoSPIM 2X magnification 3D renderings of 3-cm-long segments with HuCD-labeled neuronal cells (magenta) in the esophagus (E), duodenum (D), ileum (I), proximal colon (PC) and distal colon (DC). a-i Close-ups of longitudinal slices showing layers from serosa to lumen of the same samples imaged at high resolution with COLM. Cells in the myenteric plexus (MP) and submucosal plexus (SMP) are annotated with arrows. a-ii High-resolution maximum intensity projections showing the shape of enteric neuron plexuses. b XYZ coordinates of individual segmented cells extracted from an imaged distal colon plotted in 3D space (left) and unwrapped and flattened to be plotted as a 2D plot (right). c Quantitative analysis from 3D datasets of organs along the gastrointestinal tract measuring the volume of HuCD+ clusters (c-i) and the number of clusters (c-ii). n = 1 animal. d mesoSPIM 2X magnification 3D rendering of distal colon segment with labeled neuronal fibers (Tuj1, orange), inhibitory neurons (nNOS, yellow) and autofluorescence (cyan). d-i Single longitudinal slice imaged at high resolution with COLM. d-ii Maximum intensity projection of Tuj1+ neuron fibers and nNOS. e Longitudinal slice of distal colon (left) and result of virtual flattening the tissue to the serosa edge (right). f Plexus-specific segmentation of HuCD-labeled neurons using depth mapping of the first 50 image planes (equal to 160 μm) from the serosa edge. g Quantitative analysis of spatially separated neuronal plexuses, showing differences in the plexus’ distance from the serosa (g-i) and the percentage occupation of the tissue by the two plexuses (g-ii). n = 4 regions of interest in n = 1 animal. If not specified, scale bars are 100 μm. Quantitative data used for plotting can be found in tables within Supplementary Data 1. Abbreviations: MP myenteric plexus, SMP submucosal plexus. Statistics: Error bars are minimum and maximum. One-way ANOVA with Tukey multiple comparisons correction for (c-i) and (c-ii); Two-way ANOVA with Tukey multiple comparisons correction for (g-i) and (g-ii).
