Visualizing the molecular mode of motion from a correlative analysis of localization microscopy datasets

The determination of the mode and rapidity of motion of individual molecules within a biological sample is becoming a more and more common analysis in biophysical investigations. Single molecule tracking (SMT) techniques allow reconstructing the trajectories of individual molecules within a movie, provided that the position from one frame to the other can be correctly linked. The outcomes, however, appear to depend on the specific method used, and most techniques display a limitation to capture fast modes of motion in a crowded environment. We demonstrate here that the limitations encountered by conventional SMT can be significantly overcome by employing alternative approaches based on image spatial-temporal correlations, enabling to visually extract quantitative insights on the ensemble mode of motion of fluorescently labeled biomolecules that would otherwise be inaccessible.