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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 resolution 1. 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 …