Abstract
Understanding of the microvasculature has previously been limited by the lack of methods capable of capturing and modelling complete vascular networks. We used novel imaging and computational techniques to establish the topology of the entire blood vessel network of a murine lymph node, combining 63706 confocal images at 2 μm pixel resolution to cover a volume of 3.88 mm3. Detailed measurements including the distribution of vessel diameters, branch counts, and identification of voids were subsequently re-visualised in 3D revealing regional specialisation within the network. By focussing on critical immune microenvironments we quantified differences in their vascular topology. We further developed a morphology-based approach to identify High Endothelial Venules, key sites for lymphocyte extravasation. These data represent a comprehensive and continuous blood vessel network of an entire organ and provide benchmark measurements that will inform modelling of blood vessel networks as well as enable comparison of vascular topology in different organs.
Highlights
A novel imaging technology has been developed which extends the scale of conventional confocal microscopy beyond its depth penetration limit, enabling high-resolution imaging over large tissue volumes
We employed Extended-volume imaging system’ (EVIS) imaging to capture the full blood vascular system of a lymph node (LN) and developed computer tools designed to aid the quantification of the resulting large dataset
Most apparent is the accumulation of large vessels near the hilum, where the main artery and vein enter the LN while the outer surface comprises loops of smaller vessels (Fig. 2a,b)
Summary
A novel imaging technology has been developed which extends the scale of conventional confocal microscopy beyond its depth penetration limit, enabling high-resolution imaging over large tissue volumes. The LN vasculature reconfigures extensively during immune activation, with blood endothelial cells proliferating and the feed arteriole remodelling within 2 days after immune challenge[18,19] This vascular remodelling allows the LN to support lymphocyte accumulation, activation, and controlled expansion leading to LN swelling[20,21]. We report high-resolution imaging of the blood vessel system of an entire murine LN and introduce computational procedures for processing and analysing those images in 3D. We show how these techniques can be used to compare the vasculature of functionally different LN subregions, and determine the contribution of morphologically distinct vessels such as HEVs to the entire network This approach offers an innovative way of investigating the features of vascular networks that is applicable to a range of different tissues
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