(A) TIRF images of a Lifeact-GFP (green) expressing primary mouse B cell making a synapse with a PMS loaded with the surrogate antigen anti-Igκ F(ab’)2 (magenta), at indicated times from the initial contact. Examples of actin foci and filaments are marked by white arrows and arrowheads, respectively. Scalebar 2 μm. (B) Sideview of a Lifeact-GFP (green) expressing B cell after 20 min of contact with a PMS loaded with anti-Igκ F(ab’)2 (magenta). Scalebars 2 μm. (C) Example of actin foci and fiber segmentation in primary mouse B cells prepared as in (A). Left panel shows raw TIRF image of Lifeact-GFP, right panel shows segmented foci (magenta) and fibers (green) overlaid on the original image. Scalebar, 2 μm. (D) Number of actin foci quantified from primary B cells incubated as in (A) at the indicated times after synapse formation. Data points are mean ± SD from 310 cells in one representative experiment. (E) Distribution of the numbers of actin foci in 193 cells from a representative experiment at 20 min after synapse formation. (F) Distribution of the lifetime of actin foci in B cells from the experiment in (E). (G) Actin structure and segmentation of foci and fibers in a Ramos B cell interacting with a PMS loaded with anti-IgM. Scalebar, 2 μm. (H) dSTORM reconstruction of F-actin stained with phalloidin-AlexaFluor647 at the focal plane of a synapse using TIRF illumination of a primary mouse B cell interacting with a PMS loaded with anti-Igκ F(ab’)2. Scale bar, 2 μm. (I, J, K) Magnified areas show actin foci (arrows), filament meshwork, and lamellipodia, respectively. Gray intensity scales are different from H and are shown on the left of each image. Scale bar, 0.5 μm. (L) 3D dSTORM reconstruction of F-actin, stained with phalloidin-AlexaFluor647 (green) overlaid onto epifluorescence images of PMS loaded with anti-Igκ F(ab’)2 (antigen, magenta). Image shows a section containing actin 250–300 nm above the PMS and the corresponding epifluorescence section. Scale bar 1 μm. (M) Magnified area containing an antigen cluster lifted from the PMS, surrounded by actin foci. (N) Vertical section through this area shown as maximum intensity projection along x. Scale bars in (M), (N) 0.2 μm. (O). Histogram of foci size in the z-direction (height). Data are from 1369 foci in 58 cells from one representative experiment.