Single particle tracking in living cells: Is the third dimension worth it?

The technical advances of single particle tracking (SPT) have come with an increasing demand for such techniques to follow single biomolecules as they accomplish their function in living cells. From the single particle trajectory, quantitative mechanistic information can be obtained that are unattainable in classical ensemble methods. Traditionally, SPT is performed in two dimensions (2D) for technical simplicity. However, life occurs in three dimensions (3D) and different methods have been recently developed to track particles in 3D. Now, is the third dimension worth the effort? Here, we tracked fluorescent nanoparticles in living cells using a home-made orbital tracking microscope capable of tracking particles in 3D in real-time with a high spatial resolution1. The nanoparticles were tracked in two different cell types having different 3D aspect ratios: 3D Dictyostelium discoideum and quasi-2D HuH-7 human cells. To be compared, the 3D trajectories and their 2D projections were analyzed with a time-resolved algorithm2 based on a local mean square displacement (MSD) analysis. The distributions and characteristics of the active and passive phases were calculated in both cases. Here, we show that intra-cellular diffusion is not purely isotropic and that 2D trajectories cannot be simply scaled up to 3D. The estimation of the diffusion coefficient was more strongly biased in the HuH-7 cells whereas the active transport analysis in these quasi-2D cells was only barely affected by the projection. Inversely, a third of the active phases in the roundish amoeba were wrongly assigned to passive phases in the 2D analysis revealing the quasi isotropic organization of the cell's cytoskeleton. Hence, for an accurate determination of the diffusion coefficient and characterization of the different dynamic phases, 3D tracking and analysis are required