TCR microclusters have a reduced negative charge at the inner leaflet of the plasma membrane. (A and D) B-A8 T cells expressing either R-pre-mRFP (A) or MyrPalm-eGFP (D) probes were imaged on lipid bilayers displaying HA-DR4/ICAM-1 using TIRFM (10-s intervals, 3 min). Higher magnification indicated by inset is shown in bottom panels. Images representative of n > 3 experiments. Bar, 10 µm. (B and E) Pearson’s correlation coefficient (r) for images in A and D; results of representative images are displayed. (C and F) Fluorescence signal intensities of HA-DR4 microclusters and R-pre-mRFP were measured by connecting several microclusters using line tool (dotted lines in A and D). (G) Recruitment of ZAP-70-eGFP to synapse (10-s intervals, 3 min, TIRFM). Images representative of n = 2 experiments. Bar, 10 µm. (H) Population analysis of at least 20 cells per group for fluorophore distribution using Pearson’s correlation factor. P values were calculated using unpaired two-tailed t test with 99% CI analysis (*, < 0.0001). n > 10 cells per experiment. (I) T cells expressing R-pre-mRFP and ZAP-70-eGFP were imaged using TIRFM on negative control lipid bilayers displaying CLIP-DR4/ICAM-1. Higher magnification indicated by inset is shown in bottom panels. Images representative of n = 2 experiments. Bar, 10 µm.