Trajectory-profile-guided single molecule tracking for assignment of one-dimensional diffusion trajectories.

A variety of algorithms exist for optical single molecule tracking in two and three dimensions. One general class of algorithms employs cost-functionals to link the individual fluorescent spots, produced by a molecule in sequential video frames, into trajectories. This method has also been used to track one-dimensional (1D) molecular motions for relatively low diffusion rates (i.e., D < 1 μm2/s). At high diffusion rates, the cost-functional approach often fails to accurately reproduce 1D trajectories, particularly when the molecules are closely spaced. In this paper, we present a new algorithm called trajectory-profile-guided (TPG) tracking that is designed specifically for 1D trajectories. TPG tracking involves an initial search for one-dimensionally aligned fluorescent spots (i.e., candidate molecules). Qualifying candidates are subsequently identified and linked into trajectories based on several criteria. We test the TPG algorithm’s accuracy and precision against cost-functional based tracking using b...