Intravital three-photon microscopy allows visualization over the entire depth of mouse lymph nodes

• Type of Material: Article
• Authors: Kibaek Choe, Cornell University; Yusaku Hontani, Cornell University; Tianyu Wang, Cornell University; Eric Hebert, Cornell University; Dimitre G Ouzounov, Cornell University; Kristine Lai, Cornell University; Ankur Singh, Emory University; Wendy Béguelin, Cornell University; Ari M Melnick, Cornell University; Chris Xu, Cornell University
• Language: English
• Date: 2022-01-27
• Publisher: NATURE PORTFOLIO
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2022, The Author(s), under exclusive licence to Springer Nature America, Inc.
• Final Published Version (URL): http://dx.doi.org/10.1038/s41590-021-01101-1
• Title of Journal or Parent Work: NATURE IMMUNOLOGY
• Volume: 23
• Issue: 2
• Start Page: 330
• End Page: +
• Keywords: Life Sciences & Biomedicine; STRATEGIES; DEEP; EXPRESSION; Science & Technology; HIGH ENDOTHELIAL VENULES; IMMUNITY; IN-VIVO; FLUORESCENCE MICROSCOPY; Immunology; MOTILITY; DYNAMICS; CELL TRAFFICKING

Abstract

Intravital 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.