High affinity BCRs show an enhanced ability to form immobile oligomers. (A and B) The μ-High and μ-Low J558L cells labeled with Alexa Fluor 568–Fab anti-IgM were placed on planar lipid bilayers lacking antigen (A) or containing NIP1-His12 (B), and single BCR molecule TIRF images were acquired. (left and middle) Individual BCR molecules in TIRF images from one typical μ-High or μ-Low J558L cell (Video 1) indicating the instant diffusion coefficient (D₀) by pseudocolored spots. The display range of the pseudocolor is based on the D₀ value of 0–0.5 µm²/s, a range that included most of the D₀ values of the tracked BCRs (see E). (right) The accumulated trajectory footprints of individual BCR molecules in the entire time course. (C–H) The D₀ values for all BCR molecules from μ-High and μ-Low J558L cells (C–E) or B1-8 primary B cells (F–H) labeled with Alexa Fluor 568–Fab anti-IgM placed on planar lipid bilayers containing no antigen, NIP1-His12, or pNP1-His12. All of the D₀ values were displayed as MSD plots (C and F), CPD plots (D and G), or mean ± SD scattered plots (E and H). In C and D, the arrows indicate the change in the MSD (C) or single-molecule diffusion (D) for μ-High BCRs (red) and μ-Low BCRs (blue). Data represent single BCR molecules of the indicated numbers (D and G) for each condition from three independent experiments. The MSD plots (C and F) were further mathematically fitted into a confined diffusion model by an exponential function to acquire the size of the confinement microdomains, as detailed in Materials and methods. Significant differences by the Kolmogorov-Smirnov test are indicated (*, P < 0.0001) in D and G. One-tailed t tests were performed for statistical comparisons in E and H.