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  Toward Absolute Molecular Numbers in DNA-PAINT

Stein, J., Stehr, F., Schueler, P., Blumhardt, P., Schueder, F., Mücksch, J., et al. (2019). Toward Absolute Molecular Numbers in DNA-PAINT. Nano Letters, 19(11), 8182-8190. doi:10.1021/acs.nanolett.9b03546.

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 Creators:
Stein, Johannes1, Author              
Stehr, Florian1, Author              
Schueler, Patrick1, Author              
Blumhardt, Philipp1, Author              
Schueder, Florian2, Author              
Mücksch, Jonas1, 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: TOTAL INTERNAL-REFLECTION; SUPERRESOLUTION MICROSCOPY; LOCALIZATION MICROSCOPY; DUPLEX FORMATION; FLUORESCENCE; KINETICS; BINDING; LIMITDNA-PAINT; super-resolution microscopy; single-molecule localization microscopy (SMLM); molecular counting; fluorescence correlation spectroscopy (FCS);
 Abstract: Single-molecule localization microscopy (SMLM) has revolutionized optical microscopy, extending resolution down to the level of individual molecules. However, the actual counting of molecules relies on preliminary knowledge of the blinking behavior of individual targets or on a calibration to a reference. In particular for biological applications, great care has to be taken because a plethora of factors influence the quality and applicability of calibration-dependent approaches to count targets in localization clusters particularly in SMLM data obtained from heterogeneous samples. Here, we present localization-based fluorescence correlation spectroscopy (lbFCS) as the first absolute molecular counting approach for DNA-points accumulation for imaging in nanoscale topography (PAINT) microscopy and, to our knowledge, for SMLM in general. We demonstrate that lbFCS overcomes the limitation of previous DNA-PAINT counting and allows the quantification of target molecules independent of the localization cluster density. In accordance with the promising results of our systematic proof-of-principle study on DNA origami structures as idealized targets, lbFCS could potentially also provide quantitative access to more challenging biological targets featuring heterogeneous cluster sizes in the future.

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Language(s): eng - English
 Dates: 2019
 Publication Status: Published in print
 Pages: 9
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Degree: -

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Project name : DFG JU 2957/1-1 to R.J. SFB1032 (projects A11 and A09 to R.J. and P. Schwille)
Grant ID : -
Funding program : Emmy Noether Program
Funding organization : German Research Foundation
Project name : MolMap
Grant ID : 680241
Funding program : ERC Starting Grant
Funding organization : European Research Council

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Title: Nano Letters
  Abbreviation : Nano Lett.
Source Genre: Journal
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Publ. Info: Washington, DC : American Chemical Society
Pages: - Volume / Issue: 19 (11) Sequence Number: - Start / End Page: 8182 - 8190 Identifier: ISSN: 1530-6984
CoNE: https://pure.mpg.de/cone/journals/resource/110978984570403