Fig. 7 ∣. In vivo 3PM of T cell migration in LPS-induced inflamed LNs.

Schematics of imaging location (red box) in each LN; C, cortical side; M, medullary side in the LNs. 3D reconstruction of 650–700 μm z-stacks with the field of view of 300 × 300 μm² acquired by 1,300 nm 3PE. Naive CD8⁺ T cells and naive CD4⁺ T cells were labeled with CMRA and CFSE, respectively in LN1–2. To exclude the possible effect of labeling on results, the labeling scheme was switched for LNs 3 and 4, with CMRA and CFSE labeling CD4⁺ and CD8⁺ T cells, respectively. Four LNs from four mice were imaged. Normalized T cell distributions by depth were acquired by measuring the number of cells within a volume (300 × 300 × 50 μm³) from the indicated depth to 50 μm below. T cell mean velocity and motility coefficient were measured within a volume (300 × 300 × 100 μm³) from the indicated depth to 100 μm below. Each data point indicates a tracked cell; the number of analyzed cells (n = 3–30) 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 U-test for difference between CD8⁺ and CD4⁺ T cells.