Automatic Five-Dimensional Single Particle Tracking in Live Cells

Abstract

A novel single particle tracking (SPT) technique has been developed to visualize the intracellular transport of cargos in five dimensions (5D): the three spatial coordinates (x, y, z) and two orientation angles (azimuthal angle and polar angle) of a probe's transition dipole over a series of time steps. Tracking rotational probes, such as gold nanorods, in the z-axis is challenging because translational motions in the z-axis can be easily confused with rotational motions as both types of motion result in similar changes in signal intensity. The 5D-SPT method overcomes this long-standing challenge by utilizing the concept of parallax to sense the axial movement of the target object and employs an automatic feedback loop algorithm to control the objective's focal plane and bring the target object back to focus repetitively. In such an implementation, the target object is kept in focus to provide the highest possible S/N for 2D localization and orientation determination, while the z positions are recorded from the vertical movement of a high-precision objective scanner. Through the live-cell SPT experiments, we demonstrate that the 5D-SPT technique has potential to significantly improve the capacity to study the intracellular transport of cargos in a cellular environment.