tLNs can be repeatedly imaged in the same area days apart. C57BL/6 LNs were engrafted to the ear pinnae of hCD2-GFP mice. Three to 4 weeks after engraftment, mice were anaesthetized, and the ear pinna was mounted on a heated stage. Vasculature was visualized with nontargeted Qdots (red). Several regions of the tLN were imaged where distinctive areas of vasculature were present. Two such areas are shown (Regions 1 and 2). Mice were then recovered and returned to the animal facility. Animals were then reimaged 3 days later. The same distinct areas of vasculature could be found in the naive tLN, confirming that the same area could be imaged longitudinally. Feature blood vessels are highlighted in blue. Scale bars in Region 1 images denote 50 μm. Scale bars in Region 2 images denote 100 μm. Images were acquired with a 10×/0.3NA air objective lens. Two representative regions are displayed from the same tLN.