In this analysis, the center of mass of each molecule was tracked over consecutive images (section A.5 in Appendix).
The gap between t and t + Δ t could be closed if the probability density distribution of the relative motion of a pair of molecules is tracked using the Fokker-Planck equation [ 51, 52] (12) with the initial probability density function is (is the initial separation) and the partially absorbing boundary condition at the collision distance.
If single molecules are tracked, all three degrees of freedom have to be considered.
Single molecules were tracked and lifetimes measured using a custom Matlab script (MathWorks, Natick, MA).
The protein was injected into the cytoplasm of mouse cells, and single molecules were tracked in the nucleus with high-speed fluorescence microscopy.
However, when individual molecules were tracked with ∼10 nm precision, it was apparent that individual dynein molecules were not completely immobile in MT overlap zones, but rather frequently moved bidirectionally with long pauses between each run.
However, processive runs (4 16 steps corresponding to 144 576 nm translocation) in 0.1 2 μM ATP were observed, during which the moving molecules were tracked by manual shift of the narrow scan area.
As with our previous studies, diffusion trajectories of single fluorescent lipid and protein molecules were tracked and measured using the Particle Tracker plugin for ImageJ, yielding a per-frame quantitation of particle position and brightness.
For single molecule tracking of NTRs, the same assay was used, but Importinβ-Alexa488 at single molecule concentration was tracked with an acquisition time of 2ms on a previously described home built imaging microscope (Ori et al., 2013).
For statistical analysis of velocities of individual molecules linearly moving on a filamentous structure, the center of mass of each molecule can be tracked semi-automatically for its successive AFM images.
