ABSTRACT
Significant advances have been made in developing fluorophores and single-molecule detection techniques, allowing us to monitor single biomolecules at work in living cells directly. Significantly, the progress of single-molecule tracking/single-particle tracking (SMT/SPT) microscopy enables the visualization of the motion trajectories of individual biomolecules in living systems, revealing the molecular dynamics and transport motions of the biomolecules that support the cellular functions. Evolving from camera-based two-dimensional tracking techniques, state-of-the-art SMT/SPT microscopies developed in recent years can achieve multiparameter detections while tracking a single molecule in the 3D space. This capability has ignited a wide range of investigations at the cellular or tissue level, including identifying molecular interaction hotspots and characterizing association/dissociation kinetics between molecules. Here, we review various SMT/SPT techniques developed to date and elaborate on their potential for gaining insights into biology. In particular, we discuss the approaches to fluorescently label the target biomolecules inside the cells as the plasma membrane guard the biomolecules’ exchanges. We also share our insights about the challenges that current SMT/SPT techniques face and the potential strategies to tackle those challenges.
