Fig. 2

Voronoï-based colocalization analysis of 2-color simulation data. a 2-color 100 nm circular (purple) and square (green) clusters with inter-cluster distances of 0, 50 and 125 nm (scale bar 50 nm). Original 2-color localizations (left). Scatterplots of the normalized pairs densities for the 2 channels (middle). Scatterplots are defined for each channel, always keeping the density of channel A in abscises and the density of channel B in ordinates. The dashed line is a visual representation of the Spearman rank correlation analysis while the solid line shows the threshold used to compute the Manders coefficients. 5 class classification of the 2-color localizations computed from the overlapping Voronoï diagrams (right). b Manders’ coefficients and (c) Spearman rank correlation computed on the 3 colocalization conditions for different density ratios ranging from 1:1 (0.013 mol.nm⁻², 0.013 mol.nm^-2) to 1:5 (0.013 mol.nm^-2, 0.065 mol.nm^-2). All 3 colocalization conditions were correctly retrieved for all the densities: (d = 0 nm, M^A = 0.94 ± 0.006 SEM, M^B = 0.7 ± 0.004 SEM; d = 50 nm, M^A = 0.43 ± 0.004 SEM, M^B = 0.32 ± 0.001 SEM; d = 125 nm, M^A = 0.01 ± 0.001 SEM, M^B = 0.01 ± 0.0001 SEM) for Manders and (d = 0 nm, S^A = 0.76 ± 0.003 SEM, S^B = 0.76 ± 0.005 SEM; d = 50 nm, S^A = 0.46 ± 0.005 SEM, S^B = 0.4 ± 0.003 SEM; d = 125 nm, S^A = −0.18 ± 0.002 SEM, S^B = −0.18 ± 0.002 SEM) for Spearman rank correlation. In all box plots the center line is the median, the square is the mean and the bounds of the boxes are the 75 and 25% percentiles i.e., the interquartile range (IQR)