Choe, KibaekHontani, YusakuWang, TianyuHebert, EricOuzounov, Dimitre G.Lai, KristineSingh, AnkurBéguelin, WendyMelnick, Ari M.Xu, Chris2024-05-312024-05-312022-02K. Choe, Y. Hontani, T. Wang, E. Hebert, D.G. Ouzounov, K. Lai, A. Singh, W. Béguelin, A.M. Melnick, C. Xu. 2022. "Intravital three-photon microscopy allows visualization over the entire depth of mouse lymph nodes." Nature Immunology, Volume 23, Issue 2, pp.330-340. https://doi.org/10.1038/s41590-021-01101-11529-29081529-2916https://hdl.handle.net/2144/48938Intravital confocal microscopy and two-photon microscopy are powerful tools to explore the dynamic behavior of immune cells in mouse lymph nodes (LNs), with penetration depth of ~100 and ~300 μm, respectively. Here, we used intravital three-photon microscopy to visualize the popliteal LN through its entire depth (600-900 μm). We determined the laser average power and pulse energy that caused measurable perturbation in lymphocyte migration. Long-wavelength three-photon imaging within permissible parameters was able to image the entire LN vasculature in vivo and measure CD8+ T cells and CD4+ T cell motility in the T cell zone over the entire depth of the LN. We observed that the motility of naive CD4+ T cells in the T cell zone during lipopolysaccharide-induced inflammation was dependent on depth. As such, intravital three-photon microscopy had the potential to examine immune cell behavior in the deeper regions of the LN in vivo.p. 330-340Print-ElectronicenImmunologyBiochemistry and cell biologyAnimalsCD4-positive T-lymphocytesCD8-positive T-lymphocytesCell movementCell trackingIntravital microscopyLymph nodesMiceMicroscopy, confocalArticle2024-03-2010.1038/s41590-021-01101-10000-0002-3068-6088 (Choe, Kibaek)0000-0001-8853-9454 (Hontani, Yusaku)0000-0002-6087-6376 (Wang, Tianyu)0000-0002-1950-0668 (Lai, Kristine)0000-0002-3501-2277 (Singh, Ankur)0000-0002-4894-9229 (Béguelin, Wendy)0000-0002-8074-2287 (Melnick, Ari M)0000-0002-3493-6427 (Xu, Chris)914102