Extended Data Fig. 8: Naïve CD8⁺ and CD4⁺ T cell distribution in LNs.

a, 3D reconstruction of z-stack images (230x800x750 μm³) acquired in a popliteal LN in vivo by 3PM at 1300 nm excitation. b, Naïve eGFP⁺ CD8⁺ and DsRed⁺ CD4 T cell positions of a in yz coordinates. (bottom) Color-maps showing relative T cell density in each 100 × 100 μm² square area. c, Relative T cell distribution along the z-axis in the dashed boxes in b. Each data point represents the number of cells within the volume from the indicated depth to 50 μm below. a-c, Representative of two independent experiments. d, (1^st column) 2D-images were acquired in 50 μm cryosections of popliteal LNs by ex vivo 2PM. Naïve CD8⁺ T cell and naïve CD⁺4 T cell were labeled with eGFP and DsRed, respectively for LN1-2. The labeling scheme was switched for LN3, with DsRed and eGFP labeling CD8⁺ and CD4⁺ T cells, respectively. C, cortical side. M, medullary side. Scale bars, 200 μm. (2^nd column) Naïve CD8⁺ and CD4⁺ T cell positions in xy coordinates. Dotted circles are the area presumed to be B cell follicles. (3^rd-4^th columns) Color-maps showing relative T cell density in each 100 × 100 μm² square area.