Supplementary Figure 3: Effect of blinking on SMLM data and Ripley’s K analysis.

A molecular density of 70 molecules/µm² in ROIs of 5 × 5 µm was simulated in 10 independent runs per condition. Blinking was simulated based on experimentally determined blinking statistics (Supplementary Fig. 2). Localization maps are shown for each condition (left panels) with corresponding Ripley’s K analysis (right panels). Random molecular distributions were simulated (a) without blinking, i.e. each molecule is represented by one localization or (b) including blinking statistics determined for anti-TCRβ H57-AF647. Yellow arrows indicate clearly visible blinking-related localization clusters. (c-j) Further scenarios relevant for this study were simulated as follows: (c) Randomly distributed dimers without blinking; (d) randomly distributed dimers with blinking based on anti-CD3ε KT3-AF647; (e) randomly distributed dimers with blinking based on PS-CFP2; (f) randomly distributed dimers that diffuse according to parameters determined for the TCR (mobile fraction of 64% and D = 0.047 µm²/s) with blinking based on PS-CFP2; (g) randomly distributed monomers with blinking based on PS-CFP2; (h) a mix of 70% randomly distributed dimers and 30% randomly distributed monomers without blinking; (i) a randomly distributed mix of 70% dimers and 30% monomers that both diffuse according to parameters determined for the TCR (mobile fraction of 64% and D = 0.047 µm²/s) and that both show blinking based on PS-CFP2; (j) a randomly distributed mix of 70% dimers and 30% monomers that both show blinking based on PS-CFP2. Ripley’s K curves are shown as mean ± SD; Scale bars: 2 µm