Detection of early TCR microcluster formation by self-reactive T cells with an anti-CD3 Fab fragment. (A and B) T cells were stained with a fluorescently labeled Fab fragment specific for CD3ε and then applied to lipid bilayers containing ICAM-1 and pMHC. Cells were fixed after 5 min (A) or 30 min (B) and IRM and fluorescence images for pMHC and CD3ε were taken using TIRF microscopy. (C) Specificity of CD3ε microcluster detection by stimulation with irrelevant CLIP-MHC. Representative images at 5 min are shown. Data are representative of at least two independent experiments. Bars, 5 µm.