Extended Data Fig. 4: Single molecule TIRF fluorescence imaging analysis of pMHC molecules immobilized on 12 thermo-responsive beads at the bead/glass interface.

(a) TIRF images (left) and integrated analysis (counts of bright punctae across all images, right) for a single bead from code 8 bearing biotinylated VPLTEDALL/B35 complexes. In each image, the white circle indicates the radius of the settled bead and the orange square indicates the region of interest used to estimate pMHC density. (b) Representative TIRF images (left) and integrated analysis (counts of bright punctae across all images, right) for a single negative control bead. In each experiment, biotinylated pMHC molecules were detected by incubation with PE-labeled anti-β2 mAb (clone: A17082A) immunofluorescence. Detailed bead preparation methods are found in Methods; the orange square ROI is ~840 μm². We acquired images similar to those shown in (a) and (b) across 10 and 5 independent experiments, respectively. Scale bars: 50 µm.