Three-dimensional single-particle tracking in live cells: news from the third dimension
Single-particle tracking (SPT) is of growing importance in the biophysical community. It is used to investigate processes such as drug and gene delivery, viral uptake, intracellular trafficking or membrane-bound protein mobility. Traditionally, SPT is performed in two dimensions (2D) because of its technical simplicity. However, life occurs in three dimensions (3D) and many methods have been recently developed to track particles in 3D. Now, is the third dimension worth the effort? Here we investigate the differences between the 2D and 3D analyses of intracellular transport with the 3D development of a time-resolved mean square displacement (MSD) analysis introduced previously. The 3D trajectories, and the 2D projections, of fluorescent nanoparticles were obtained with an orbital tracking microscope in two different cell types: in Dictyostelium discoideum ameba and in adherent, more flattened HuH-7 human cells. As expected from the different 3D organization of both cells' cytoskeletons, a third of the active transport was lost upon projection in the ameba whereas the identification of the active phases was barely affected in the HuH-7 cells. In both cell types, we found intracellular diffusion to be anisotropic and the diffusion coefficient values derived from the 2D analysis were therefore biased.