Intravital three-photon microscopy of entire mouse lymph node

Abstract For the last 20 years, intravital confocal and two-photon microscopy have been powerful tools to explore dynamic immune cell behavior in mouse lymph node. However, they can only image ~100 and ~300 μm depth respectively while a peripheral lymph node of adult mouse has 600 – 1000 μm thickness. Here, we visualized whole thickness of adult mouse popliteal lymph node with intravital three-photon microscopy. Three-photon excitation significantly improved a signal-to-background ratio compared to two-photon excitation even at the same excitation wavelength. The capability to image the full depth enabled to achieve the 3D volume of entire lymph node vasculature in vivo by tiling multiple z-stack images. We observed DsRed-expressing lymphocyte migration in LYVE1+ lymphatic sinus at 600 μm depth of lymph node where conventional two-photon microscopy was normally inaccessible. In addition, we demonstrated the capability multi-color imaging by using Cγ1-Confetti mice of which each germinal center B cells stochastically expresses one of 4 different fluorescent proteins (CFP, GFP, YFP and RFP). Intravital three-photon microscopy has the potential to shed light on unknown immune cell behavior in deeper regions of the lymph node in vivo just as two-photon microscopy did during the last 20 years at the shallower depth.