FcγRs reorganize into concentric rings upon activation. (A) TIRF images of FcγRI (top) and FcγRII (bottom) at the surface of human macrophages incubated for 10 or 30 min on slides coated with PLL (nonactivated) or hIgG and stained with fluorescently labeled specific antibodies. Bars, 10 µm. (B) TIRF and dSTORM images of FcγRI (green) and FcγRII (red) at the surface of macrophages incubated for 10 or 30 min on slides coated with PLL or hIgG and stained with anti–FcγRI-AF488 and anti–FcγRII-AF647 mAbs. Bars, 5 µm. Regions outlined by the white squares (middle column) are shown enlarged (right column) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (C) CBC histograms of the single-molecule distributions of the colocalization parameter for FcγRI and FcγRII in cells seeded onto PLL- or hIgG-coated slides for 10 (light gray and dark gray, respectively) or 30 min (light red and dark red, respectively) or for positive control data (green). The positive control data in this figure are the same as in Fig. 2. Data are from a minimum of 10 cells from three independent donors. Bars represent mean ± SD. (D) NND analysis from data shown in C. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P < 0.01; ****, P < 0.0001; one-way ANOVA with Tukey’s post-hoc test. (E) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥20,000 clusters from a minimum of 10 cells per condition). a.u., arbitrary units; NN, nearest neighbor; PC, positive control.