Fig. 6 ∣. Measurement of T cell motility across the entire depth of popliteal LNs.

Schematics of imaging location (red box) in each LN; C, cortical side; M, medullary side in the LNs. 3D reconstruction of 620–680 μm z-stacks with the field of view of 404 × 404 μm² acquired by 1,300 nm 3PE. Naive CD8⁺ T cells and naive CD4⁺ T cells were labeled with DsRed and eGFP, respectively, in LN1–3. To exclude the possible effect of the labeling on the results, the labeling scheme was switched for LNs 4 and 5, with DsRed and eGFP labeling CD4⁺ and CD8⁺ T cells, respectively. Five LNs from five mice were imaged. Normalized T cell distributions by depth were acquired by measuring the number of cells within a volume (202 × 202 × 50 μm³) from the indicated depth to 50 μm below at the center of the 3D z-stacks. T cell mean velocity and motility coefficients were measured within a volume (202 × 202 × 35 μm³) from the indicated depth to 35 μm below at the center of the 3D z-stacks. Each data point indicates a tracked cell; the number of analyzed cells (n = 11–30) is indicated on the graphs; the median with the interquartile range; Kruskal–Wallis test followed by Dunn’s multiple comparisons test for differences between depths; two-tailed Mann–Whitney test for difference between CD8⁺ and CD4⁺ T cells.