Figure 5 |. Astigmatism-based 3D tokPAINT enables axial antibody mapping throughout cryosections.

a Validation of cylindrical lens addition to a commercial TIRF system for 3D DNA-PAINT imaging. Left: Astigmatism-based encoding of axial position by reshaping the point spread function. Right: Sum DNA-PAINT 467 DNA origami with 20 nm docking strand pattern. b Left, top and middle: x and y line plot histograms across docking strand position indicated by white dashed circle in (a). Left, bottom: Axial distribution of z coordinates of DNA origami data set. The standard deviation obtained by a Gaussian fit (red curve) is given above the histograms. Right: correction of axial sample tilt affecting measured z-distributions (red dashed curve in c). c LeZ: 3D tokPAINT of Pol II S5p. The color code indicates axial position of antibody signal over a range of 150 nm. Right: 3D Exchange-tokPAINT image of Lamin A/C (green), Pol II S5p (blue) and SC35 (orange). Side view (x-z) of localization clusters projected from white box. Scale bars, 5 μm in (b), 3 μm in (c) and 150 nm in zoom-in.