Fig. 2: Structure of PCVs and their branching networks.

a–d, Maximum projection images of deep two-photon imaging stacks showing PCVs in a 6-month-old adult mouse (a,b) and a 22-month-old aged mouse (c,d). e,f, Schematic of vessel types and their nomenclature in PCV networks. e, Structure of PCV drainage network. f, Anatomy of PCV branch networks. g, Side projection of deep in vivo two-photon imaging stack showing a PCV in an adult mouse, overlaid with Imaris reconstructions of PCV branch vascular networks in layers 2/3, 4 and 6/CC. h–j, Top-down views of Imaris reconstructions of PCV branch vascular networks in layers 2/3 (h), 4 (i) and 6/CC (j). The red circles correspond to the point where the main branch joins the trunk of the PCV. The gray-colored vessels correspond to pre-convergence capillaries, violet-colored vessels represent other tributary vessels and cyan-colored segments correspond to the main branch segments. k,l, Three-photon imaging to define the gray–white matter boundary. Side projections of image stacks showing the 3HG signal fluorescence from myelin (k) and the vasculature labeled with the FITC-dextran intravenous dye (l) in an awake adult mouse (aged 6 months). The transition from gray to white matter is visible in the 3HG signal image (white dotted line). m, Maximum projection image showing a top-down view of the layer 6/CC branch (750–1,000 µm depth) from l. The white matter is a striated 3HG signal coming from axon bundles of the CC. n, Side view of reconstructed PCV branch in m. The vascular segments draining layer 6 and the CC are labeled in cyan and yellow, respectively. The dotted white line marks the location of the gray–white matter transition.