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  Flat-top TIRF illumination boosts DNA-PAINT imaging and quantification

Stehr, F., Stein, J., Schueder, F., Schwille, P., & Jungmann, R. (2019). Flat-top TIRF illumination boosts DNA-PAINT imaging and quantification. Nature Communications, 10: 1268. doi:10.1038/s41467-019-09064-6.

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 Creators:
Stehr, Florian1, Author              
Stein, Johannes1, Author              
Schueder, Florian2, Author              
Schwille, Petra1, Author              
Jungmann, Ralf2, 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: SUPERRESOLUTION MICROSCOPY; LOCALIZATION MICROSCOPY; PERFORMANCE; BINDINGScience & Technology - Other Topics;
 Abstract: Super-resolution (SR) techniques have extended the optical resolution down to a few nanometers. However, quantitative treatment of SR data remains challenging due to its complex dependence on a manifold of experimental parameters. Among the different SR variants, DNA-PAINT is relatively straightforward to implement, since it achieves the necessary 'blinking' without the use of rather complex optical or chemical activation schemes. However, it still suffers from image and quantification artifacts caused by inhomogeneous optical excitation. Here we demonstrate that several experimental challenges can be alleviated by introducing a segment-wise analysis approach and ultimately overcome by implementing a flat-top illumination profile for TIRF microscopy using a commercially-available beam-shaping device. The improvements with regards to homogeneous spatial resolution and precise kinetic information over the whole field-of-view were quantitatively assayed using DNA origami and cell samples. Our findings open the door to high-throughput DNA-PAINT studies with thus far unprecedented accuracy for quantitative data interpretation.

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Language(s): eng - English
 Dates: 2019
 Publication Status: Published online
 Pages: 8
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 Table of Contents: -
 Rev. Type: -
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Project name : (DFG JU 2957/1-1 to R.J.)
Grant ID : -
Funding program : Emmy Noether Program
Funding organization : German Research Foundation
Project name : ERC Starting Grant (MolMap)
Grant ID : 680241
Funding program : -
Funding organization : European Research Council

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Title: Nature Communications
  Abbreviation : Nat. Commun.
Source Genre: Journal
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Publ. Info: London : Nature Publishing Group
Pages: - Volume / Issue: 10 Sequence Number: 1268 Start / End Page: - Identifier: ISSN: 2041-1723
CoNE: https://pure.mpg.de/cone/journals/resource/2041-1723