A guide to single-particle tracking

Individual proteins and protein complexes undergo various motion types, including free diffusion, confinement, subdiffusion and directed motion. Different motion behaviours reflect different microenvironments, activity states, kinetics and interaction partners. Single-particle tracking (SPT) is a powerful method for analysing these behaviours directly and in live cells. However, SPT is confounded by multiple sources of experimental noise and biases. Interpreting tracks in terms of quantitative models thus remains a challenging task. We start this Primer by briefly presenting experimental setups and labelling techniques often employed for SPT, followed by a focus on the variety of tools available for analysing noisy tracks with multiple states. This includes tools designed to identify and characterize state fractions and diffusion coefficients, detect and quantify state transitions, predict the number of states and identify and parameterize various motion behaviours. We then highlight some of the many applications of SPT in cellular biology and discuss the limitations of current methods and what future developments are needed to address the current challenges of the SPT analysis.