Figure 4. Spiders imaged on origami tracks in real-time using super-resolution TIRF microscopy.

a, Position-time trajectory of a selected spider (EAC 2, Cy3-labeled) on the EAC substrate track. The position as a function of time is represented by color-coded dots (see Supplementary Information for details). A small green dot represents the START and a large red oval represents the Cy5-labeled STOP site. ZnSO₄ was added at time zero. b, Displacement of the spider trajectory in panel a from its initial position as a function of time. The green line represents displacement calculated using averaged position measurements of 1 min intervals, and the black line represents the displacement from a rolling 4-min average (see Supplementary Information). c, Ensemble root mean square displacement (RMSD) of exemplary spiders on the EAC substrate track in the presence (red, corresponding to the 15 Tier 1 Spiders in Supplementary Fig. 29) and absence (black, 7 spiders) of Zn²⁺, with the corresponding displacements used to calculate each ensemble RMSD for each buffer condition (similarly colored line graphs). d, Ensemble RMSD for spiders on EAC tracks satisfying simple filtering criteria. Curves are shown for spiders on EAC substrate track (red, 85 spiders), EAC product track with TRIGGER introduced to the sample 10-15 min before imaging (blue, 18 spiders), and EAC product track with TRIGGER introduced 30-60 min before imaging (black, 29 spiders). EAC substrate and 10-15 min trigger product RMSD plots are fit to a power law function, and the EAC 30-60 min trigger product RMSD is fit to a straight line. Individual displacements are shown with colors corresponding to the respective ensemble RMSD plots. All Figure 4 data were obtained in SSC buffer.