Supplementary Figure 6: Sensitivity of label-density-variation SMLM to detect nanoclustering in experiments using KT3-AF647 (a) and PS-CFP2 (b).

Normalized ρ versus η plots were calculated for different simulated clustering scenarios and assessed for the difference from simulated random molecular distributions; detectable difference (dark gray), borderline (light gray) and not detectable difference (white) (see Supplementary Fig. 5a). Reference numbers indicate scenarios published in the literature. Simulations of nanoclusters with radii of 20, 40, 60, 80, 100 and 150 nm for 3, 5, 10, 15 and 20 clusters/µm². The fraction of molecules inside clusters was varied between 40% and 100%. Average molecular densities were adjusted to (a) 70 molecules/µm² based on KT3-AF647 labeling of CD3ε (Supplementary Fig. 5a) or (b) 76 molecules/µm² based on ζ-PS-CFP2 PALM experiments (Fig. 3a). Blinking statistics were based on experimental data of the respective fluorescent probes. Numbers in boxes indicate the average number of molecules per cluster and the relative enrichment of molecules inside versus outside of clusters is indicated. The relative clustered area (right) was calculated from thresholded binary cluster maps. b is an extension of Fig. 4b: panels for 20 nm and 60 nm are identical to Fig. 4b