Intravital three-photon microscopy of T-cell migration in the T-cell zones over the entire depth of mouse popliteal lymph node

Abstract Most T cells in a popliteal lymph node are located in T-cell zones at ~150 to ~600 μm below the cortical surface of the lymph node. For the last 20 years, intravital two-photon microscopy has been a powerful tool for observing the three dimensional T-cell migration in the T-cell zones. However, two-photon imaging can only visualize superficial T-cell zones to ~300 μm depth. In this work, we successfully visualized T-cell migration in T-cell zones over the entire depth of a mouse popliteal lymph node with intravital three-photon microscopy. We measured the change in lymphocyte velocity with increasing laser powers at 600 μm depth to determine the safe laser power range for visualizing T-cell migration. We found that, with less than 80 mW at 1300 nm excitation, the migration of EGFP-expressing lymphocytes were not perturbed by the laser illumination. Using the safe power of 1300 nm excitation, we observed dynamic migration of EGFP-expressing naïve CD4 T cells and DsRed-expressing naïve CD8 T cells simultaneously in T-cell zones at various depths. The naïve T cells were sparse at 0 ~150 μm depth in cortical side (follicular and interfollicular regions), dense in the T-cell zones, and less dense in the medulla, which was identified by labeling with eFluor615-conjugated LYVE-1 antibody in vivo. We found that the density distributions of CD4 and CD8 T cells were different in the T-cell zones. We also measured the migration velocity and motility coefficient for naïve CD4 and CD8 T cells in the T-cell zones over the entire depth of the lymph node. This work shows that three-photon microscopy has the potential to shed light on immune cell dynamics in the deep regions of lymph nodes that are typically inaccessible to conventional two-photon microscopy.