High-resolution 3D imaging and topological mapping of the lymph node conduit system
EVIS imaging of an entire popliteal LN generated a 3D volume image of which interior slices can be viewed individually (A). Three-dimensional image reconstruction of the entire LN shows (B) lymphatic channels filled with the tracer molecule WGA (red glow) together with dextran-labeled blood vessels (green), or the blood vasculature alone (C). An interior view of 20-μm thick optical sections (D—I) and a 1 μm slice (J) permits detailed insights into the LN architecture. A cross-section of the LN displays the location of cell-specific zones (D), and close-ups reveal anatomical details, including the arrangement of long conduits descending from the SCS at the edges of a B cell follicle (arrowheads, E), an MC with a central blood vessel situated amongst the WGA-filled sinuses of the medulla (F), and the transition from dense to sparse conduit networks in the superficial to the deep TCZ (G). The conduit network forms a highly organized grid within the TCZ (white, H and I; red, J) interspersed with CS (arrowhead, panel J) and blood vessels (green), including HEVs, which are closely surrounded by cells displaying a cobblestone-like morphology (arrowhead, panels H and J). Besides larger blood vessels, small blood vessels are frequently enclosed by conduits (asterisks, G and J). Image rendering was performed in Voxx (A—I) and ImageJ (J). See also S1 Fig, S1 and S2 Videos. CS, cortical sinus; EVIS, extended-volume imaging system; HEV, high endothelial venule; LN, lymph node; MC, medullary cord; SCS, subcapsular sinus; TCZ, T-cell zone; WGA, wheat germ agglutinin.