The upper panels show data accumulated by TIRF imaging a single Cy3B molecule dried onto a glass coverslip. (A) The sum of 1000 raw image frames recorded with an exposure time of 0.08 s per frame. B shows the output after Gaussian fitting, whereas image C is the sum of the frames with values for Δx below 4.80 nm (solid line in Fig. 1),;the scale bars in A, B, and C are 100 nm. The lower panels show TIRF images of quantum dot aggregates dried onto a glass coverslip composed of 5000 summed frames recorded with an exposure time of 0.08s in D the raw diffraction limited format, E after Gaussian fitting and F with a Lucky threshold of 9.4 nm applied to the localization data. The scale bars are 250 nm in these three images. The panel to the far right shows the look-up table for the images ranging, from black at low intensity to white at high intensity.