Figure 1.

AnaDDA. (A) The effect of transition rates on D^∗ distributions is depicted with simulated tracks of four steps and different transition rates. With increasing transition rates relative to the frame rate, the bound and unbound distributions start merging toward an intermediate apparent diffusion speed diffusivity (right). The distribution of apparent diffusion coefficients D^∗, calculated from the mean jump distances of a track, originates from the finite number of steps (n) that are measured for each particle and allows the extraction of the underlying diffusion coefficient and transition kinetics of the states. (B) Procedure of anaDDA is as follows: the D^∗ values from tracked single particles are run into an MLE optimization program that refines a set of start parameters based on the likelihood to find a certain value given the number of steps (all tracks longer than eight are reduced to the first eight steps). (C) Shown is a comparison of simulated (gray bars) and theoretically predicted (black line) distribution with different amount of tracks and the following starting parameters: $k_{\text{on}}^{\ast }$ = 0.2 frame⁻¹, $k_{\text{on}}^{\ast }$ = 0.2 frame⁻¹, D_free = 4 μm²/s, σ = 30 nm (localization precision), and step number = 4 steps. Tracks are simulated without any confinement boundaries. The Kolmogorov-Smirnov test statistic (D_KS) is indicated at each histogram. (D) Shown is the Kolmogorov-Smirnov test statistic compared with the threshold for statistically distinguishable distributions. Values above the threshold line indicate that two distributions significantly differ from each other. Error bars indicate SEM of three independent simulations. (E) Shown is a comparison of simulated D^∗ distributions (gray) and the distributions calculated with anaDDA for different transition rates (black). The shape of the distributions depends on both the ratio between $k_{\text{on}}^{\ast }$ and k_off and the absolute values of these parameters. In this example, D_free = 4 μm²/s, and σ = 30 nm. For more tested parameters, see Fig. S1. To see this figure in color, go online.