SIRPα and FcγRI are arranged in discrete nanoclusters at macrophage surfaces. (A and B) TIRF and dSTORM images of SIRPα (A) and FcγRI (B) at the surface of human macrophages seeded onto PLL- (nonactivated, top) or hIgG-coated slides (bottom) for 10 min and stained with fluorescently labeled specific antibodies. Bars, 5 µm. Regions delineated by white squares are zoomed-in and shown with corresponding density maps (pseudocolor scale), thresholded binary maps and Ripley’s K analysis of the molecules in the selected regions. Bars, 1 µm. L(r)-r represents the degree of clustering relative to simulated random distributions, indicated by the 99% confidence intervals (CIs); r is the radial scale. (C–E) Nanocluster areas (C), density (D), and percentage of localizations in nanoclusters (E) for SIRPα and FcγRI under nonactivating (black) or hIgG-activating (gray) conditions were calculated by subjecting dSTORM data to spatial point-pattern analysis and thresholding. Each symbol represents the median of several 5 × 5 µm regions from the same cell. Horizontal lines and error bars represent mean ± SD. Data are from a minimum of 30 cells from three independent donors. ns, not significant; ****, P < 0.0001; two-tailed t test assuming unequal variance. (F and G) Label-density variation analysis for SIRPα (F) and FcγRI (G) yields characteristic normalized ρ/η curves for clustered proteins. Cells were stained with anti–SIRPα-AF647 (F) or anti–FcγRI-AF488 (G) at different labeling concentrations and imaged by dSTORM. Each data point represents a single cell, color-coded by antibody concentration used for labeling. Red lines indicate reference curves for a random distribution of molecules.