Fig. 5. Large-scale molecular phenotyping-compatible 3D imaging of cerebral vasculature.

a Maximum projection of light-sheet fluorescence microscopic images of the entire vasculature in a SeeNet-treated brain (1.6× zoom, ×0.63 objective lens, numerical aperture: 0.15, working distance: 87 mm) into a single stacked photo. Casted vessels are shown in red. Arterioles immunolabeled with anti-αSMA are shown in green. Scale bar = 1 mm. b The RITC–Dex–GMA signal in a, shown on a grayscale, was optically sectioned at Z = 3 mm. Scale bar = 1 mm. c, d Magnified images of the boxed region of b. Vasculature in the gray matter c and white matter d was visualized with capillary resolution. Scale bar = 200 μm. e The RITC–Dex–GMA signal in a posterior part of the left hemisphere in a is shown on a grayscale. Magenta indicates cortico-hippocampal vascular paths observed in this area. Scale bar = 1 mm. See also Supplementary Movie 3. f The anti-αSMA immunosignal of the boxed regions in e is shown on a green scale. Magenta contours indicate cortico-hippocampal vascular paths. Each end of the vascular path is denoted by CTX (cortical side) or HPC (hippocampal side). Arteriole–arteriole (f₁), venule–arteriole (f₂), and venule–venule (f₃) connections were observed. Scale bar = 100 μm. See also Supplementary Movie 4. g The percentage of the connected pairs of dorsal hippocampal transverse arterioles (red) and venules (blue) to the total number of vessels observed. h Maximum projection of confocal images of casted vasculatures from the pia to the hippocampus. A cortico-hippocampal vascular path is shown in magenta. Scale bar = 100 μm. See also Supplementary Movie 5. i The anti-αSMA immunosignal in the same field is shown on a green scale. Scale bar = 100 μm. See also Supplementary Movie 6. j A 3D-rendered view of the cortico-hippocampal vascular path. Source data are provided as a Source Data file.