Fig. 1: BTS diagram to investigate oligomerisation and diffusion dynamics.

a Cartoon sketching the sFCS acquisition in the plane of an SLB (left) and changes in biomolecular organisation (right). Increase in diffusion coefficient (magenta) causes faster molecular movement, increase in oligomerisation causes higher molecular brightness. b Hypothetical BTS diagram demonstrating fast monomeric diffusion (left) versus slowed-down oligomeric diffusion (right). c, d Computer-simulations of freely diffusing molecules sampled by sFCS. Change in brightness (c to d) shifts the distributions on the y-axis and changes in diffusion coefficient (as indicated on panels) shifts distribution on x-axes. 10 measurements yielding 500 curves per condition were simulated. e, f Assessment of sFCS raw data quality for the simulations of free diffusion across the parameter space. Better data is represented by a low nRMSD value or a high SNR value, respectively. g, h Computer-simulations of binary mixtures (monomers and dimers g and monomers and trimers h) with increasing fraction of molecules in an oligomerised state (as indicated). Number of molecules in the simulation was kept constant and 500 curves were simulated per condition. i,j sFCS raw data quality for the simulations of oligmerisation given by nRMSD and SNR evaluation. k Average brightness from the simulations on oligomerisation (monomers plus either dimers, trimers or tetramers) as described before (fraction 0.5, solid lines represent averages and standard deviations of the mean values from 10 simulations with 500 autocorrelation curves total). For the four sets of simulations the brightness values were significantly different from each other with p < 0.01 (one-way ANOVA with Tukey’s test for multiple comparisons). l–n Changes in average transit time and brightness with changing fraction of oligomerisation for monomers plus dimers, trimers or tetramers, respectively. Dots represent averages and error bars standard deviations from 10 simulations including 500 curves. The brightness and transit time values for all fractions ≥0.1 were statistically significantly different from the monomer condition (i.e., at oligomer fraction=0.0, p < 0.01; two-way ANOVA with Tukey’s test for multiple comparisons).