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. 2020 Jul 27;10(1):12504.
doi: 10.1038/s41598-020-68892-5.

Three-dimensional super-resolution fluorescence imaging of DNA

Sevim Yardimci  1 , Daniel R Burnham  1 , Samantha Y A Terry  2 , Hasan Yardimci  3
Affiliations

Three-dimensional super-resolution fluorescence imaging of DNA

Sevim Yardimci et al. Sci Rep. .

Abstract

Recent advances in fluorescence super-resolution microscopy are providing important insights into details of cellular structures. To acquire three dimensional (3D) super-resolution images of DNA, we combined binding activated localization microscopy (BALM) using fluorescent double-stranded DNA intercalators and optical astigmatism. We quantitatively establish the advantage of bis- over mono-intercalators before demonstrating the approach by visualizing single DNA molecules stretched between microspheres at various heights. Finally, the approach is applied to the more complex environment of intact and damaged metaphase chromosomes, unravelling their structural features.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
YOYO-1 and SxO binding and unbinding kinetics in different buffers. (a)–(d) Time-lapse measurements of association kinetics at 20 nM YOYO-1 (a) or SxO (b). The chemical structure of YOYO-1 is shown while that of SxO is proprietary information. Dissociation kinetics of YOYO-1 (c) and SxO (d) under three different buffer conditions. Buffer conditions are TE50 (10 mM Tris pH 8.0, 1 mM EDTA, 50 mM NaCl), TE50 containing either Ascorbic Acid and Methyl Viologen (ROXS) or glucose, glucose oxidase, catalase and MEA-HCl (IB) with concentrations indicated in the methods section. The overlaid lines are fits to the data using the model equations described in the methods section.
Figure 2
Figure 2
3D BALM imaging of DNA. (a) A cartoon depiction of a DNA-coated bead immobilized on a glass surface through biotin-streptavidin linkage. (b, c) 3D BALM images (b) and the corresponding cross sections of two DNA-coated microspheres at different axial levels (c) with color-coded height map. The schematics above c illustrate different z levels. (d)–(f) 3D BALM images of 10 kb linear dsDNA tethered to the surface of the glass at both ends (d), tethered to the surface at one end and to a 1-µm diameter bead at the other end (e), tethered to a 1-µm diameter bead at one end and to a 0.4-µm diameter bead at the other end (f). (g) Line profile for determination of lateral and axial FWHM (representing dsDNA, tethered to a 1-µm diameter bead from one end and to a 0.4-µm diameter bead from the other end). Solids lines are fits to a Gaussian.
Figure 3
Figure 3
3D super-resolution imaging of metaphase chromosomes. (a) Three-dimensional BALM images of an undamaged human metaphase chromosome. (b) 3D BALM images of an irradiated chromosome. A schematic representation of irradiated chromosome with a groove on one of the arms and three-dimensional surface plots at the upper (blue), and lower (red), layers. (c) (Top left) Wide-field image of a SxO-stained human chromosome (blue) and telomeric regions on the chromosome (magenta). (Bottom left) A top view of merged re-constituted super resolution (BALM) image of the chromosome and re-constituted super resolution (STORM) image of the telomeric regions of this chromosome. (Right) Three-dimensional surface plots of BALM (blue), STORM (red), and both images superimposed.
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