High-throughput super-resolution single-particle trajectory analysis reconstructs organelle dynamics and membrane reorganization.

Super-resolution imaging can generate thousands of single-particle trajectories. These data can potentially reconstruct subcellular organization and dynamics, as well as measure disease-linked changes. However, computational methods that can derive quantitative information from such massive datasets are currently lacking. We present data analysis and algorithms that are broadly applicable to reveal local binding and trafficking interactions and organization of dynamic subcellular sites. We applied this analysis to the endoplasmic reticulum and neuronal membrane. The method is based on spatiotemporal segmentation that explores data at multiple levels and detects the architecture and boundaries of high-density regions in areas measuring hundreds of nanometers. By connecting dense regions, we reconstructed the network topology of the endoplasmic reticulum (ER), as well as molecular flow redistribution and the local space explored by trajectories. The presented methods are available as an ImageJ plugin that can be applied to large datasets of overlapping trajectories offering a standard of single-particle trajectory (SPT) metrics.