Extended Data Fig. 2 ∣. Lymphocyte velocity at 600 μm (a) and 300 μm depth (b) with increasing average power of 1300 nm excitation.

a-b, eGFP⁺ lymphocytes were imaged at the same site with 4 different average powers by 3PE at 1300 nm. The average power (Power) at surface is proportional to the pulse repetition rate (PRR) while the pulse energy (Pulse E) at focus remains approximately constant. For each depth, four LNs from 3 mice were imaged. The exact range of imaging depths around the nominal imaging depths of 600 μm (a) and 300 μm (b) were 590 μm to 625 μm and 290 μm to 325 μm, respectively. The average power increases with the depth from top (Z1) to bottom (Z2) of the imaging volume. Effective attenuation length (EAL) was calculated by taking 4 images at different depths (150, 300, 450, 600 μm depths for a, 50, 100, 200, 300 μm depths for b). Each data point indicates an individual lymphocyte track; n = 30 tracks for each condition; the median with the interquartile range; ns, not significant; Kruskal-Wallis test followed by Dunn’s multiple comparisons test.