Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes

Single-particle tracking methods allow detailed analysis of protein movement in cells, but existing tracking algorithms have substantial limitations, particularly at high particle densities. A new software tool overcomes some of these limitations and can be used to track high-density particles in cell membranes. Also in this issue, Jaqaman et al . describe an alternative software tool for high-density single-particle tracking. Although the highly dynamic and mosaic organization of the plasma membrane is well-recognized, depicting a resolved, global view of this organization remains challenging. We present an analytical single-particle tracking (SPT) method and tool, multiple-target tracing (MTT), that takes advantage of the high spatial resolution provided by single-fluorophore sensitivity. MTT can be used to generate dynamic maps at high densities of tracked particles, thereby providing global representation of molecular dynamics in cell membranes. Deflation by subtracting detected peaks allows detection of lower-intensity peaks. We exhaustively detected particles using MTT, with performance reaching theoretical limits, and then reconnected trajectories integrating the statistical information from past trajectories. We demonstrate the potential of this method by applying it to the epidermal growth factor receptor (EGFR) labeled with quantum dots (Qdots), in the plasma membrane of live cells. We anticipate the use of MTT to explore molecular dynamics and interactions at the cell membrane.