Figure 2

Multiplexed Exchange-PAINT super-resolution imaging of fixed BT20 cells. (a–e) Five-“colour” receptor images. (Top) Schematic of imaging procedure. (Bottom) Exchange-PAINT images. Each receptor target is labelled with an antibody carrying a unique Exchange-PAINT docking strand. Imaging is performed sequentially using five orthogonal imager strands (a^*, b^*, c^*, d^*, e^*) all at 2.5 nM and labelled with the fluorophore ATTO655. (f,g) Merged images of all five targets for (f) unstimulated or (g) EGF-stimulated cells. Inset panels (labelled i to viii) 10-fold higher magnification views of regions of the images in f and g that highlight receptor clusters with varied shapes and compositions. Imaging: 15,000 frames per cycle at 10 Hz rate. Washing: 1–2 minutes per cycle. (h,i) Ripley’s K-function analysis of multiple regions selected for unstimulated (h) and EGF-stimulated cells (i). Mean L(r)—r reports the degree of clustering for several regions in a population of cells relative to a random distribution (indicated by the grey line), and r indicates the radius. Scale bars in a–g: 5 µm; scale bars for the insets (labelled i-viii) is 1 µm.