High-resolution 3D imaging and topological mapping of the lymph node conduit system
Differences in conduit density between the deep and superficial TCZ can be visualized by averaging and color-coding pixel densities over small image volumes in a “moving average” display, shown as a rainbow spectrum (A, B). A cross-section of the moving average display exposes how dense regions in the periphery of the LN (blue) surround an inner region of lower conduit density (green), directly representing dense or sparse occurrence of conduit segments in the corresponding section of the conduit network image (dark blue, left panel), respectively (A). Volume rendering (B) of the entire TCZ using this approach shows the dense superficial zone (blue) enclosing a central region of sparse conduits (green). Alternatively, the TCZ conduit map was employed for calculating the distances to the nearest conduit, and voids with a distance of over 8 μm were displayed in red, indicating larger distances within the deep TCZ as opposed to outer regions (C). From these 2 zones, 10 subregions were selected for comparative analysis (D), including the number of vertices (E), the number of conduit segments (F), the conduit volume (G), conduit segment diameters (H), conduit segment lengths (I), and the combined conduit length (J). The distributions of the minimum distances to the nearest conduit (K) and the average minimum distance (L) further exemplify the larger spacing within the deep TCZ. Simulation of T-cell motility predicts similar motility coefficients within the deep TCZ and the surrounding superficial zone (M). Data are from 1 experiment (each point represents one 100 μm subregion, N = 10) and plots show means ± SD. ****p < 0.0001, ***p < 0.001, ns = not significant, Student t test. Values for each data point can be found in S1 Data. See also S4 Fig. LN, lymph node; TCZ, T-cell zone.