Rapid TCR clustering and internalization during the initial stages of antigen recognition. CMTMR-labeled OT-I–GFP Rag2^−/− cells were transferred into WT recipients and immunized as in Fig. 5. LNs were explanted and visualized by two-photon microscopy. Data are representative of more than five experiments. For all data with antigen, time = 0 represents the time at which the antigen was added. (a, b, and d) Maximum intensity z projections of OT-I–GFP fluorescence represented in pseudocolor (top) and overlaid OT-I–GFP fluorescence in green and CMTMR fluorescence in red (bottom). (a) Representative time-lapse image of OT-I–GFP TCR localization on a crawling T cell in a nondraining LN in the absence of antigen. White arrowheads indicate the leading edge of the cell of interest. Image was collected using the 510/20-nm filter. (b) Representative time lapse of sustained OT-I–GFP TCR clustering after antigen addition. Image was collected using the 525/50-nm filter. For full video, see Video 6. (c) TCR clustering associated with the cell surface. Orthogonal views of OT-I–GFP fluorescence of the T cell represented in b showing a 1.6 (x) × 1.6 (y) × 2–µm (z) slice through the cell at the location indicated by the crosshairs. (d) Representative time lapse of OT-I–GFP TCR internalization after antigen addition. Image was collected using the 525/50-nm filter. For full video, see Video 7. (e) TCR internalization. Orthogonal views of OT-I–GFP fluorescence of the T cell represented in d showing a 1.6 (x) × 1.6 (y) × 2–µm (z) slice through the cell at the location indicated by the crosshairs. Bars, 10 µm.