Fig. 4: Localization precision and resolution in MINSTED nanoscopy.

a, Localization precision (points: median; shaded areas: ±1 and ±2 standard deviations) measured from many consecutive binding events on clustered binding sites (blue) on DNA origami grids of 12-nm periodicity and for each binding event individually (red). Blue points and shades are displayed only if computed from at least ten clusters. The red solid line shows the estimated localization precision for the individual events; resulting from that, instabilities are considered to reconstruct the cluster data (blue solid line). Simulated localizations without background are shown as the red dashed line. b, MINSTED image of rectangular binding site pattern of 12-nm periodicity and the pertinent localization distribution in c. Each localization is represented by its estimated position. Blue circles correspond to 1 (solid) and 2 (dashed) standard deviations of the estimated binding site position. The cluster identity is color-coded. d, MINSTED image of 3 × 3 hexagonal DNA origami with internal distances of 6 nm. e, Overlay of 59 MINSTED images of the origami pattern from d, completely resolving the periodically arranged bindings of 6-nm mutual distance. The data were filtered according to Supplementary Table 1. f, Binding sites of 4-nm distance are fully resolved by MINSTED; sketch of the underlying origami design is shown below. The circles of 2-nm diameter represent the extent of the Cy3B molecules whose structure is drawn to scale (upper-right corner) to highlight the relationship between the localization precision and the fluorophore size.