Fig. 3: Localization precision and artifacts.

a, Distribution of 3D position measurements for single Alexa Fluor 647 molecules, determined using the dynamic spline PSF model. The size of the distribution provides a direct measure of the localization precision of the microscope. On average, 8,002 photons were detected per fluorophore switching event. b, A 3D Gaussian fit to the histogram of the localization distribution yielded a standard deviation of 3.4 nm in the x and y dimensions, and 2.1 nm in the z dimension, corresponding to a spatial resolution with full width at half maximum values of 8 nm laterally and 5 nm axially. c, Variation of the localization precision as a function of the z coordinate of the emitter. By stepping a fluorescent bead through the focus of the microscope, the precision was measured over a 2.2-µm range. The bead brightness was similar to a single fluorophore (9,200 photons per frame). The CRLB, which estimates the theoretical limit of the precision on the basis of the information content of the data, is also shown (solid lines). d, Fraction of localizations erroneously assigned to the wrong interference fringe by the fitting procedure, as a function of the z coordinate of the emitter. When such an error occurs, the z position of the emitter is apparently shifted by a multiple of the PSF fringe spacing (λ/2), and this results in periodic ghost artifacts in the final image. Over a central range of 1.5 µm, no localization artifacts were observed.