Supplementary Figure 5: Classification criteria for cluster detection and sensitivity of label-density-variation SMLM to detect nanoclustering in experiments using H57-AF647.

(a) Schematic normalized ρ versus η plots of random (red) and different cluster scenarios (blue): cluster scenarios were classified as clearly detectable, when confidence intervals did not show overlap at η = 0.3 (left); as borderline, when confidence intervals overlapped at η = 0.3, but mean values were outside the confidence intervals (center); as not detectable, when mean values were lying within the respective confidence intervals at η = 0.3 (right). All curves are plotted as mean ± SEM. The dashed line indicates η = 0.3, where the detectability was evaluated. (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) as shown in a. 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 73 molecules/µm² based on H57-AF647 labeling of TCRβ (Fig. 2a). Blinking statistics were based on experimental data of H57-AF647. 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. The figure is an extension of Fig. 4a: panels for 20 nm and 60 nm are identical to Fig. 4a