Tracking and inference of complete MT plus-end trajectories. (A) Sub-tracks, which are generated by linking +TIP comets and which indicate the growth of MTs, are unidirectional and often collinear (arrow pairs). Pairs of collinear growth sub-tracks may become candidates for sub-track linking. Bar, 5μm. (B) Illustration of the candidate selection for sub-track linking. Sub-track i (black) starts at t = 0 and ends at t = 8. Candidate sub-tracks j (blue and green) for linking to sub-track i must initiate in the gray search region and start within Δtmax frames (user input to the tracking software) from the end-point of sub-track i. Candidates chosen from the light gray region will generate inferred shrinkage events (blue), while candidates chosen from the dark gray region will generate inferred pause or continuation-of-growth events (green; the latter arises due to detection failure or the comet moving temporarily out of focus). The cost of linking depends on the time gap between the sub-track end and candidate start, Δtgap, and the three spatial parameters d||, d⊥, θ. See text for details of these parameters and the cost function. (C) The cost of sub-track linking is directionally unbiased, as shown by the distribution of costs in the forward (green) and backward (blue) directions for all potential links (left). For tracks with only one candidate, the balance is maintained (right). Sub-track pairs with costs higher than the death cost (vertical line) will not be chosen for linking, allowing proper termination of trajectories where pause or shrinkage is unlikely. (D) The ‘Track Overlays’ tool () was used to show all tracks on the image (left; bar, 10μm), zoom in, select an individual track (right; bar, 5μm) from the hundreds shown in the inset, and view its profile (table). In this example profile the lifetimes and displacements are reported in seconds and microns, while the software returns them in frames and pixels. This track corresponds to . (E) Evaluation of frame-to-frame and sub-track linking (forward and backward gaps separated) in simulated +TIP movies.