Simulation of SPT experiments to investigate the permeability of the actin barrier. (a) Schematic representation of the model geometry that was used. The crossing probability (cp) is a measure of the likelihood that a particle will be able to pass through the actin barrier. (b) Single-molecule localization reconstructions were generated using Fluosim from simulations of particles moving randomly over the model geometry in (a). 10 reconstructions per cp were used to determine the mean peak at 200 nm of the autocorrelation function. Error bars indicate standard deviation. (c) Exemplary reconstructions of simulated SPT experiments for varying cp’s. Note that domains become apparent at cp = 0.7. Very low cp leads to confinement of molecules, prohibiting the exploration of the geometry. (d) Autocorrelation function (ACF) of reconstruction described in (a) for cp = 1. Since actin is fully permeable, no peaks in the autocorrelation function can be detected. (e) Like (d), but for cp = 0.7. A periodicity of ∼200 nm is clearly apparent in all reconstructions. (f) Like (d), but for cp = 0.2. Scale bar is 500 nm.