Single-particle tracking (SPT) offers insights into dynamics, interactions, and organization of living biological samples by analyzing the motion of individual particles. SPT determines the localization of individual molecules as a function of time yielding information on various subpopulations beyond the average. Single localization points are merged into trajectories (figure 1b). Calculation of the mean square displacement (MSD) (figure 1c) for individual trajectories allows the determination of several parameters, such as diffusion coefficients (figure 1d), confinement radii, and diffusion types (figure 1e), i.e. whether particles are immobile, confined, or freely diffusing.
As low densities of fluorophores are needed to differentiate between single particles, a combination of super-resolution techniques and SPT is advantageous. sptPALM uses photoactivatable fluorescent proteins which are activated, localized, and bleached rapidly. The method can be applied in observations both of cell membranes, e.g. for the observation of membrane receptors, and of intracellular processes. A related technique to discern single diffusing molecules is universal point accumulation for imaging in nanoscale topography (uPAINT). It uses fluorescently labeled structures which reversibly bind to the biological target, e.g. fluorescently labeled ligands binding to a membrane receptor. By applying TIRF illumination, fluorophores are only excited during such a binding event. For both sptPALM and uPAINT, signals of individual fluorophores are temporarily differentiated and localized to reconstruct single particle trajectories.
Figure 1: Single-particle tracking of membrane receptors. (a) Model of membrane receptors bound by fluorescently-labeled ligands moving on different trajectories on a cell membrane. (b) Recorded trajectories of MET receptors in a living HeLa cell. Color scale shows variation in calculated diffusion coefficients (scale bar 5 µm). (c) Distribution of diffusion coefficents can be calculated by analyzing single-particle trajectories. (d) Each diffusing particle is classified according to its diffusion type as immobile, confined or freely diffusing.