Intravital three-photon microscopy allows visualization over the entire depth of mouse lymph nodes
Date
2022-02
Authors
Choe, Kibaek
Hontani, Yusaku
Wang, Tianyu
Hebert, Eric
Ouzounov, Dimitre G.
Lai, Kristine
Singh, Ankur
Béguelin, Wendy
Melnick, Ari M.
Xu, Chris
Version
Accepted manuscript
OA Version
Citation
K. 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/s41590-021-01101-1
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.