The growth in FI of BCR microclusters is antigen affinity dependent. (A–F) The normalized FI of BCR microclusters examined by Igα-YFP (A and D) and Alexa Fluor 568–Fab anti-IgM (B and E) or of antigen microclusters examined by NIP1-His12-Hylight647 (C and F) are given with time (Video 8) for μ-High or μ-Low J558L cells placed on planar lipid bilayers containing NIP1-His12 (A, B, D, and E) or NIP1-His12-Hylight647 (C and F) at a concentration of 10 nM (25 molecules/µm²) or 50 nM (100 molecules/µm²), as indicated. (G–I) The normalized FIs of Cy3–Fab anti–MHC I (G), IgM–Alexa Fluor 488 (H), and NIP1-His12-Hyligh647 (or pNP1-His12-Hyligh647; I) microclusters are given for B1-8 primary B cells placed on planar lipid bilayers containing the high affinity antigen NIP1-His12 or the low affinity antigen pNP1-His12. In A–I, the data represent means ± SEM of indicated numbers of microclusters in three independent experiments. (J and K) Also given are pseudocolor 2.5D Gaussian images of typical antigen microclusters examined by NIP1-His12-Hylight647 at the indicated times for μ-High (J) or μ-Low (K) J558L cells placed on antigen-containing lipid bilayers. The FWHM of each microcluster upon 2D Gaussian fitting was used as a measure of the size of the microclusters, as detailed in Materials and methods and Fig. S4 A. Bars, 1.5 µm.