Supplementary Figure 7: Characterization of single molecule signals in STED microscopy and simulated STED microscopy data of different clustering scenarios.
From: TCRs are randomly distributed on the plasma membrane of resting antigen-experienced T cells
Characterization of single molecule signals in STED microscopy: (a) Fitting of single molecule signals with a Gaussian intensity profile yielded the integrated intensity (I) and the width (σ). Values for I could be fitted well with a log-normal distribution (dashed line), yielding a mean of 35.14 and a variance of 508.52. (b) The signal width σ showed a characteristic dependence on the intensity. The data were fitted with Eq. 1 (black line). Simulated STED microscopy images (left) analyzed by image autocorrelation analysis. Red curves represent analysis of simulated random data (n = 5); blue curves represent analysis of simulated cluster scenarios (n = 5). Molecular densities were matched to the experiment. (c) A random distribution of molecules. (d-j) Different cluster scenarios characterized by cluster radius (r), fraction of molecules inside clusters and density of clusters per area (clusters/µm²): (d) r = 20 nm, 80% in clusters, 20 clusters/µm²; (e) r = 20 nm, 100% in clusters, 10 clusters/µm²; (f) r = 60 nm, 100% in clusters, 10 clusters/µm²; (g) r = 20 nm, 100% in clusters, 20 clusters/µm²; (h) r = 60 nm, 100% in clusters, 20 clusters/µm²; (i) r = 100 nm, 15% in clusters, 0.5 clusters/µm²; (j) r = 40 nm, 10% in clusters, 0.5 clusters/µm². Curves are shown as means ± SEM; Scale bars: 500 nm
