At the Movies: 3-Dimensional Technology and Gastrointestinal Histology Ya–Yuan Fu, Shiue–Cheng Tang  Gastroenterology  Volume 139, Issue 4, Pages 1100-1105.e2 (October 2010) DOI: 10.1053/j.gastro.2010.08.025 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 (A, B) Opaque and optical-cleared mouse stomachs. The transparent stomach allowed luminal contents to be seen inside. Bar = 1 cm. (C, D) Transmitted light micrographs of the optical-cleared mouse duodenum, showing the interior domains of the villi and crypts. Arrows indicate the microvessels with a diameter of 5–10 μm. Bar = 50 μm. (E, F) 3D projections of the villus microstructure acquired from autofluorescence imaging by confocal microscopy. The 2 projections were derived from the same image stack. In (E), a cuboid was digitally subtracted to reveal the interior domain of the scanned volume. In (F), an orthogonal view was used to present the villus microstructure. A continuous display of the orthogonal views is shown in Supplementary Video 1. Dimensions of the scanned volume: 354 (X) × 354 (Y) × 245 μm (Z; depth). Gastroenterology 2010 139, 1100-1105.e2DOI: (10.1053/j.gastro.2010.08.025) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 (A) High-resolution, 3D confocal microscopy of the mouse ileal villi and crypts. Propidium iodide and carbocyanine dye DiD were used to label the nuclei (orange) and membranes (gray), respectively. Cuboids were digitally subtracted to reveal the interior domains of the scanned volumes. (B) 3D projection of the ileal vasculature. Vessel painting and nuclear staining were used to simultaneously reveal the vasculature (cyan) and nuclei (gray) in 3D confocal microscopy. A fly-through presentation of (B) is shown in Supplementary Video 2. Gastroenterology 2010 139, 1100-1105.e2DOI: (10.1053/j.gastro.2010.08.025) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 TH staining of the islet/pancreatic sympathetic innervation. Two types of TH staining are seen in (A–G) and (H–N); only the latter shows the TH-positive cell bodies in the islet. Both patterns, on the other hand, show that the TH-positive nerve fibers are associated with the blood vessels surrounding the islet. Green and gray signals are from the TH and propidium iodide staining, respectively. (A–D) and (H–K) Standard 2D confocal micrographs at different depths in the islet. (E, L) and (F, M) 3D projections of the merged TH and propidium iodide signals and the separate TH signals, respectively. (G, N) 3D projection of the TH signals on top of a 2D image section to outline the location(s) of islet(s). (A–G) and (H–N) Images derived from the stacks with dimensions of 521 (X) × 521 (Y) × 150 μm (Z) and 521 (X) × 521 (Y) × 201 μm (Z), respectively. Arrows and circles indicate the locations of the islets. Z indicates the depth of the image section relative to the top of the scanned volume. Gastroenterology 2010 139, 1100-1105.e2DOI: (10.1053/j.gastro.2010.08.025) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1 Gross views (A, B) and high-resolution (C, D) images of the pancreatic TH staining (green). Additional signals are from the nuclei (red; propidium iodide staining) and recombinant GFP (PGK-1–driven, universal GFP expression in the transgenic mouse; gray) to illustrate the pancreatic microstructure. Dimensions of the imaged volume: 921 (X) × 921 (Y) × 250 μm (Z, depth) of (A) and 1300 (X) × 1300 (Y) × 185 μm (Z) of (B). The coordinates were added by using the “Local Axis” function of the Amira software. In both (A, B), signals of the nuclei and GFP were digitally subtracted from the top areas to expose the projections of the sympathetic nerve fibers and the embedded islets (3 in panel A and 4 in panel B, as indicated by the arrows). (C, D) High-resolution, 3D images of the TH staining for visualization of the islet sympathetic innervation. (C) The dimensions of the imaged volume are 369 (X) × 369 (Y) × 222 μm (Z; depth). (D) A zoomed-in, 3D presentation of (C) showing the enlarged features of the TH-positive endocrine cell bodies and the sympathetic nerve fibers. A cuboid of propidium iodide and GFP signals was digitally subtracted from the scanned region to reveal of the TH signals. Gastroenterology 2010 139, 1100-1105.e2DOI: (10.1053/j.gastro.2010.08.025) Copyright © 2010 AGA Institute Terms and Conditions