Motility of T cells during antigen recognition. CMTMR-labeled OT-I–GFP Rag2^−/− cells and CFP⁺ polyclonal CD8 T cells were cotransferred into CD11c-YFP recipients, and recipients were immunized with CFA in the footpads. 18 h after T cell transfer and immunization, popliteal LNs were explanted and visualized by two-photon microscopy before and after the addition of SL8 peptide antigen. Imaging was performed in the most superficial 80 µm of the LN to replicate imaging conditions during TCR visualization. Data were pooled from four experiments. See Video 5 for representative video. For all data with antigen, time = 0 represents the time at which the antigen was added. (a) Representative tracks displaying cell paths during 10 min of imaging. (b) Mean T cell crawling velocity. (c) Duration of T cell arrest. (d) Duration of T cell–DC contact. (b–d) Statistical analysis was performed using a one-way analysis of variance with Tukey’s post test. Each dot represents one cell, and the bars represent the mean.