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

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 similar to 100 and similar to 300 mu m, respectively. Here, we used intravital three-photon microscopy to visualize the popliteal LN through its entire depth (600-900 mu m). 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.