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

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

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molecules-23-03165-v4.pdf (Publisher version), 5MB
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Raw data for DNA-PAINT, SI-FCS and confocal FCS measurements.

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 Creators:
Blumhardt, Philipp1, Author              
Stein, Johannes1, Author              
Mücksch, Jonas1, Author              
Stehr, Florian1, Author              
Bauer, Julian1, Author              
Jungmann, Ralf2, Author              
Schwille, Petra1, Author              
Affiliations:
1Schwille, Petra / Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, Max Planck Society, ou_1565169              
2Jungmann, Ralf / Molecular Imaging and Bionanotechnology, Max Planck Institute of Biochemistry, Max Planck Society, ou_2149679              

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Free keywords: SINGLE-MOLECULE FLUORESCENCE; OXYGEN SCAVENGING SYSTEM; TOTAL INTERNAL-REFLECTION; SUPERRESOLUTION MICROSCOPY; LOCALIZATION MICROSCOPY; OXIDATIVE DAMAGE; KINETICS; ACID; MECHANISMS; DIFFUSIONBiochemistry & Molecular Biology; Chemistry; DNA-PAINT; surface-integrated fluorescence correlation spectroscopy (SI-FCS); reactive oxygen species; photo-induced DNA damage; super-resolution microscopy;
 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.

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Language(s): eng - English
 Dates: 2018
 Publication Status: Published online
 Pages: 27
 Publishing info: -
 Table of Contents: This article belongs to the Special Issue Single-Molecule Fluorescence Spectroscopy
 Rev. Type: -
 Identifiers: ISI: 000454523000115
DOI: 10.3390/molecules23123165
 Degree: -

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Title: Molecules
Source Genre: Journal
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Publ. Info: Basel : MDPI
Pages: - Volume / Issue: 23 (12) Sequence Number: 3165 Start / End Page: - Identifier: ISSN: 1420-3049
CoNE: https://pure.mpg.de/cone/journals/resource/954925623244