Effects of geometrical confinement and the length of the tracking window. (A) Typical confinement shapes within cells are shown. The boundary shape of spherical cells is defined by a single parameter (radius; rconf.), whereas rod-shaped cells are defined by two parameters (radius and length; rconf and lconf). (B and C) Shown is the influence of spherical- and rod-shaped boundaries on the distribution of simulated (gray box) and uncorrected DDA (gray line) and corrected DDA (black line) distributions (koff = 0.2 frame−1, = 0.2 frame−1, step number = 4 steps). (D and E) Shown is the influence of spherical- and rod-shaped cells on the estimation of parameters of DDA on unconfined simulated trajectories (yellow), uncorrected DDA on confined simulated trajectories (orange), and corrected DDA on confined trajectories (red). (F and G) Shown is the same as (B–E) except for simulated trajectories with a maximal step size. Simulation parameters are as follows: Dfree = 4 μm2/s, σ = 30 nm (localization precision), and n = 50,000 tracks. The Kolmogorov-Smirnov test statistic (DKS) is indicated in each histogram. To see this figure in color, go online.