Fig. 5: ISM-FLUX on DNA-origami nanorulers.

a–c ISM-FLUX on DNA-origami with 40 nm spacing between nearest neighbors. a Different representations of the same data set show the on-off blinking and the signal on the detector for a representative on-event. b Histogram of the experimental localization uncertainty of all blinking events and the sum of all micro-images during the on-events. It shows a segment of a 9.6-min ISM-FLUX measurement. c Scatter plot of the localizations (top-left). Only localizations that pass through all filters are shown. The background image shows the sums of all MDFs. The colored scatter plot (right) shows a zoomed-in region, with the three docking sites of each origami shown in a different color. Classification based on k-means clustering with nine clusters. Reconstructed images made with a Gaussian kernel with a fixed standard deviation of 6 nm. The single-element detector plot (bottom-left) shows the localizations obtained by summing the signal over all detector elements, thus ignoring the spatial information on the detection side. d, e Data are similar to (b, c) for a DNA-origami sample with 20 nm spacing between nearest neighbors. The final localization uncertainty, calculated as the square root of the average variance in x and y for all localizations within a cluster, are (7.1 ± 1.1) nm and (4.9 ± 0.4) nm for the nanorulers with 40 nm spacing and 20 nm spacing, respectively