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Molecules 2018, 23(12), 3165; https://doi.org/10.3390/molecules23123165

Photo-Induced Depletion of Binding Sites in DNA-PAINT Microscopy

1
Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany
2
Molecular Imaging and Bionanotechnology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany
3
Department of Physics and Center for Nanoscience, Ludwig Maximilian University, Geschwister-Scholl-Platz 1, 80539 Munich, Germany
*
Author to whom correspondence should be addressed.
Academic Editor: Jörg Fitter
Received: 31 October 2018 / Revised: 26 November 2018 / Accepted: 29 November 2018 / Published: 30 November 2018
(This article belongs to the Special Issue Single-Molecule Fluorescence Spectroscopy)
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Abstract

The limited photon budget of fluorescent dyes is the main limitation for localization precision in localization-based super-resolution microscopy. Points accumulation for imaging in nanoscale topography (PAINT)-based techniques use the reversible binding of fluorophores and can sample a single binding site multiple times, thus elegantly circumventing the photon budget limitation. With DNA-based PAINT (DNA-PAINT), resolutions down to a few nanometers have been reached on DNA-origami nanostructures. However, for long acquisition times, we find a photo-induced depletion of binding sites in DNA-PAINT microscopy that ultimately limits the quality of the rendered images. Here we systematically investigate the loss of binding sites in DNA-PAINT imaging and support the observations with measurements of DNA hybridization kinetics via surface-integrated fluorescence correlation spectroscopy (SI-FCS). We do not only show that the depletion of binding sites is clearly photo-induced, but also provide evidence that it is mainly caused by dye-induced generation of reactive oxygen species (ROS). We evaluate two possible strategies to reduce the depletion of binding sites: By addition of oxygen scavenging reagents, and by the positioning of the fluorescent dye at a larger distance from the binding site. View Full-Text
Keywords: DNA-PAINT; surface-integrated fluorescence correlation spectroscopy (SI-FCS); reactive oxygen species; photo-induced DNA damage; super-resolution microscopy DNA-PAINT; surface-integrated fluorescence correlation spectroscopy (SI-FCS); reactive oxygen species; photo-induced DNA damage; super-resolution microscopy
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Blumhardt, P.; Stein, J.; Mücksch, J.; Stehr, F.; Bauer, J.; Jungmann, R.; Schwille, P. Photo-Induced Depletion of Binding Sites in DNA-PAINT Microscopy. Molecules 2018, 23, 3165.

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