BCR signaling induces a transient increase in BCR diffusion followed by receptor immobilization. (A) Dual View TIRFM to simultaneously visualize ezrin-GFP (grayscale) and track single molecules of IgM in Hel-specific A20 B cells either unstimulated or during early (<5 min) and later (>15 min) times after settling on Hel-coated coverslips. Tracks are color-coded based on their diffusion coefficient to highlight slow diffusing IgM: red < 0.01 µm² s⁻¹ and yellow > 0.01 µm² s⁻¹. Images in the bottom row are the zoomed regions indicated by white boxes. Ag, antigen. Bars, 2 µm. (B) Diffusion coefficients of single molecules of IgM in unstimulated and at early (<5 min) and late (>15 min) time points after stimulation with immobilized Hel, with the median indicated under the x axis and by the red bars. ***, P < 0.0001. (C) Cumulative probability plots for the data shown in B, in which a shift to the right or the left indicates increased and decreased diffusion, respectively. (D) Diffusion coefficients of single molecules of IgM in Hel-specific A20 B cells either unstimulated or at early (<5 min) and late (>15 min) time points after stimulation with anti-IgG, with the median indicated under the x axis and by the red bars. **, P = 0.0012. (E) Cumulative probability plots for the data shown in E. Data are representative of at least two independent experiments.