Resolving cargo-motor-track interactions with bifocal parallax single-particle tracking.

Resolving coordinated biomolecular interactions in living cellular environments is vital for understanding the mechanisms of molecular nanomachines. The conventional approach relies on localizing and tracking target biomolecules and/or subcellular organelles labeled with imaging probes. However, it is challenging to gain information on rotational dynamics, which can be more indicative of the work done by molecular motors and their dynamic binding status. Herein, a bifocal parallax single-particle tracking method using half-plane point spread functions has been developed to resolve the full-range azimuth angle (0-360°), polar angle, and three-dimensional (3D) displacement in real time under complex living cell conditions. Using this method, quantitative rotational and translational motion of the cargo in a 3D cell cytoskeleton was obtained. Not only were well-known active intracellular transport and free diffusion observed, but new interactions (tight attachment and tethered rotation) were also discovered for better interpretation of the dynamics of cargo-motor-track interactions at various types of microtubule intersections.