Extended Data Fig. 4: Lymphocyte velocity at 600 μm depth with increasing average power and pulse energy at 1650 nm excitation.

a-f, DsRed⁺ lymphocytes were imaged at the same site with 3 to 4 different pulse repetition rates by 3PE at 1650 nm. The average power at surface (Power) is proportional to the pulse repetition rate (PRR) while the pulse energy at focus (Pulse E) remains constant. Six LNs from 4 mice were imaged. g-i, DsRed⁺ lymphocytes velocity was measured at the same site with 4 different pulse energies (at focus). The average power is proportional to the pulse energy while the repetition rate was kept constant (0.33 MHz). Three LNs from 3 mice were imaged. a-i, The exact imaging depth was from 590 μm to 625 μm. The average power increases with depth from top (Z1) to bottom (Z2) of the imaging volume. Effective attenuation length (EAL) was calculated by taking 4 images at different depths. Each data point indicates an individual lymphocyte track; the number of analyzed tracks (n = 21–37) is indicated on the graphs; the median with the interquartile range; ns, not significant; Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Even though most lymphocytes stopped migration at >40mW in a,b and e (Supplementary Movie 3), the measured mean velocity is non-zero due to the uncertainties that occur when determining the cell positions at different times.

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