Digitally Assisted Single-Particle Tracking for Accurate Analysis of Complicated Cargo Transport Dynamics in Microtubule Networks.

Intracellular transport is a fundamental process crucial for cellular function, driven by the coordinated action of motor proteins that move cargo along microtubule tracks. Traditional tracking methods primarily focus on cargo trajectories, often overlooking rotational dynamics and their impact on cargo interactions with the complex microtubule network. To address this limitation, we introduced a digitally assisted single-particle tracking (dSPT) method that significantly advances the angular resolution of intracellular cargo dynamics. By integrating intensity measurements with advanced digital classification algorithms to process defocused half-plane image patterns captured through bifocal parallax microscopy, this approach extends the angular resolution range from the conventional method to a full 0-360° range, even in heterogeneous cellular environments, while maintaining high spatial and temporal resolutions. In intracellular transport events, we directly observed the accurate determination of the chiral rotational directions and precise calculation of the step angles. When combined with super-resolution radial fluctuation (SRRF) imaging to achieve higher-resolution microtubule imaging, our dSPT technique enables in vivo investigations of cargo dynamics during intracellular transport. To validate this, we studied the rotational dynamics of the cargo in microtubule confinement. Furthermore, we identified characteristic patch-searching patterns in the microtubule network, where cargo exhibited a combined motion pattern of confined and hopping diffusion to navigate through the constraints imposed by the microtubules.