Published on Sep. 14, 2020

Single molecule tracking (SMT) is a technique of single-molecule fluorescence imaging that allows for the exploration of molecular motion at a high spatiotemporal resolution on living cells. This is widely used to define dynamics of individual tumor cell-surface receptors. Spatiotemporal regulation of many of these receptors varies across cancer types, playing a key role in tumor progression and drug resistance. Many tools can be used to identify trajectories and calculate their features from these experiments. However, there are relatively few tools to analyze this data. Thus, the present study uses a set of live-cell single-molecule imaging experiments with a model tumor cell-surface receptor (EGFR) to develop a custom R-based platform to visualize and analyze single-molecule trajectories. The first analysis uses two-dimensional kernel density estimates to create a density map of molecular coordinates. The second set of analyses returns spatial heat maps based on trajectory coordinates. The final set of analyses uses high-resolution molecular coordinates to identify colocalized receptors. These analyses are fully customizable, easily visualized, and generalizable across data extraction tools and biological variables.