Precise Detection and Visualization of Nanoscale Temporal Confinement in Single-Molecule Tracking Analysis

The plasma membrane consists of a diverse mixture of molecules that dynamically assem-ble into a highly non-random organization. The formation of nanoscale domains in the membrane is of particular interest as these domains underlie critical cellular functions. Single-molecule tracking is a powerful method to detect and quantify molecular motion at high temporal and spatial resolution and has therefore been instrumental in understanding mechanisms that underlie membrane organization. In single-molecule trajectories, regions of temporal confinement can be determined that might reveal interesting biophysical interactions important for domain formation. How-ever, analytical methods for the detection of temporal confinement in single-molecule trajectories depend on a variety of parameters that heavily depend on experimental factors and the influence of these factors on the performance of confinement detection are not well understood. Here, we present elaborate confinement analyses on simulated random walks and trajectories that display transient confined behavior to optimize the parameters for different experimental conditions. Further-more, we demonstrate a heatmap visualization tool that allows spatial mapping of confinement hotspots relative to subcellular markers. Using these optimized tools, we reliably detected subdif-fusive behavior of different membrane components and observed differences in the confinement behavior of two types of glutamate receptors in neurons. This study will help in further understanding the dynamic behavior of the complex membrane and its role in cellular functioning.