Dissociation of the CD3ε cytoplasmic domain from the plasma membrane after TCR stimulation with peptide-MHC. T cells expressing the CD3ε-YF-TFP probe were stimulated with HA-DR4/ICAM-1 lipid beads. Shortly after bead engagement, cells were labeled with R18 in cold PBS and imaged to acquire FLIM data. Beads forming synapses are indicated (*). All data are representative of n > 3 experiments. (A) Confocal image of CD3ε-YF-TFP–expressing T cells forming synapses with HA-DR4/ICAM-1 lipid beads. Local accumulation of CD3ε is observed at bead–cell interface. Bar, 10 µm. (B and D) Cells with significant accumulation of CD3ε-YF-TFP probe at synapse and uniform R18 labeling were used for quantification. Right panel shows zoomed area of inset. FLIM measurements were taken at synapse and nonsynapse membrane (ROI delineated by white and red dotted lines, respectively). Bar, 10 µm. (C and E) Relative distribution of mean TFP fluorescence lifetime per pixel during FLIM-FRET within and outside synapse (blue and red line, respectively), compared with basal FLIM (black line). (F) TFP fluorescence decay curves at synapse (blue) and outside synapse (red, other PM). (G) Population analysis of FLIM measurements inside or outside of synapse (syn), compared with basal FLIM before R18 labeling; error bars represent SEM. P-values were calculated using an unpaired two-tailed Student’s t test with a 99% CI (*, >0.0001; **, 0.152). n > 5–10 cells per experiment and condition.