SPT reveals complex VIF motility. (A) (Top) Sparse SunTag labeling of the vimentin cytoskeleton in an RPE cell. (Bottom) Pseudocolored maximum intensity time projection of vimentin-SunTag puncta over 60 s. (B) SPT trajectories of vimentin-SunTag spots in a single cell over 1 min. Trajectories have been color-coded based on maximum displacement. The magnified inset is shown at right. (C) Representative vimentin-SunTag trajectories grouped by motile behavior. Numbers indicate track duration (in frames), dots indicate track starting points, edges are colorized by relative track position, and arrowheads indicate final track positions. (D) Representative vimentin-SunTag trajectory with multiple pausing events indicated by arrows. (E) Net displacement of the trajectory in E over time. Pausing events can be detected as extended durations with negligible changes in net displacement. (F) Representative vimentin-SunTag trajectory color-coded by the alpha value derived from sliding window MSD analysis (20 frame window width). (G) Alpha values for each 20-frame window centered at the indicated frame. (H) Frequency distribution of maximum displacements for standardized, 30-frame vimentin-SunTag trajectories indicating a cutoff used for immotile (<1 µm, blue) or motile (>1 µm, red) classification. N = 884 trajectories from the representative cell in B. (I) Distribution of directionality ratios for trajectories in each motility group. (J) MSD analysis of each motility group displayed on log–log axes. (K) Velocity autocorrelation at the first lag (0.5 s) for each motility group.