Application of conventional MPT analysis to simulated and experimental particle trajectory data. (A,C) Deff distribution and (B,D) ensemble geometric average MSD based on simulated or experimentally measured particle traces, respectively. In (A,C), the vertical dashed line indicates the cutoff between strongly hindered and mobile particles (see Methods). In (B,D), expected MSD values are based on simulation parameters (B) or the geometric average of MSDs for PS-COOH and PS-PEG particles imaged separately (D). Error bars represent standard error across 3 independent data sets or mucus samples.