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Volume 23
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10.3390/molecules23123165
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Article

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

by
Philipp Blumhardt
1,
Johannes Stein
1,
Jonas Mücksch
1,
Florian Stehr
1,
Julian Bauer
1,
Ralf Jungmann
2,3 and
Petra Schwille
1,*
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.
Molecules 2018, 23(12), 3165; https://doi.org/10.3390/molecules23123165
Submission 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.
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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MDPI and ACS Style

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. https://doi.org/10.3390/molecules23123165

AMA Style

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(12):3165. https://doi.org/10.3390/molecules23123165

Chicago/Turabian Style

Blumhardt, Philipp, Johannes Stein, Jonas Mücksch, Florian Stehr, Julian Bauer, Ralf Jungmann, and Petra Schwille. 2018. "Photo-Induced Depletion of Binding Sites in DNA-PAINT Microscopy" Molecules 23, no. 12: 3165. https://doi.org/10.3390/molecules23123165

APA Style

Blumhardt, P., Stein, J., Mücksch, J., Stehr, F., Bauer, J., Jungmann, R., & Schwille, P. (2018). Photo-Induced Depletion of Binding Sites in DNA-PAINT Microscopy. Molecules, 23(12), 3165. https://doi.org/10.3390/molecules23123165

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