Flat-top TIRF illumination boosts DNA-PAINT imaging and quantification View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2019-12

AUTHORS

Florian Stehr, Johannes Stein, Florian Schueder, Petra Schwille, Ralf Jungmann

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. More... »

PAGES

1268

References to SciGraph publications

Identifiers

URI

http://scigraph.springernature.com/pub.10.1038/s41467-019-09064-6

DOI

http://dx.doi.org/10.1038/s41467-019-09064-6

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1112879275

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/30894522


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51 schema:description 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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