MPLSM of tLN. 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 throughout the tLN was clearly visible after intravenous injection of nontargeted Qdots (red); GFP⁺ cells (green); second harmonic signal (blue). (A) Extended focus images of 60-μm z-stacks showing 2 different regions of the same tLN in left and right panels (scale bars, 55 μm and 50 μm, respectively). (B left panel) Three-dimensional reconstruction of blood vessels in the tLN. (B right) The corresponding extended focus image is shown; scale bar, 50 μm. (C left) Three-dimensional reconstruction of a third tLN showing vasculature. (C right) A representative optical section from the same z-stack is shown; scale bar, 43 μm. Images were acquired with a 20×/1.0NA water-immersion objective lens. Representative images from 3 independent animals are shown.