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 (A). The tLN was then imaged by MPLSM without the need for surgical exposure (ie, through the intact ear). Recirculating GFP⁺ T cells in the tLN were imaged for ≤ 30 minutes (B), and cells were tracked (C) to allow calculation of velocity (D), meandering index (E), and displacement rate (F). After imaging the intact tLN, a surgical incision was made proximal to the engraftment area. After a period of 40 minutes, the tLN was imaged again, representing the LN after injury. Excised popliteal LNs from hCD2-GFP mice were imaged in a perfusion chamber as a control. Data represent tracks generated from 3 independent animals. Scale bars represent 40 μm. ***P < .001.