Protocol | Published:

Super-resolution microscopy with DNA-PAINT

Nature Protocols volume 12, pages 11981228 (2017) | Download Citation

Abstract

Super-resolution techniques have begun to transform biological and biomedical research by allowing researchers to observe structures well below the classic diffraction limit of light. DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) offers an easy-to-implement approach to localization-based super-resolution microscopy, owing to the use of DNA probes. In DNA-PAINT, transient binding of short dye-labeled ('imager') oligonucleotides to their complementary target ('docking') strands creates the necessary 'blinking' to enable stochastic super-resolution microscopy. Using the programmability and specificity of DNA molecules as imaging and labeling probes allows researchers to decouple blinking from dye photophysics, alleviating limitations of current super-resolution techniques, making them compatible with virtually any single-molecule-compatible dye. Recent developments in DNA-PAINT have enabled spectrally unlimited multiplexing, precise molecule counting and ultra-high, molecular-scale (sub-5-nm) spatial resolution, reaching 1-nm localization precision. DNA-PAINT can be applied to a multitude of in vitro and cellular applications by linking docking strands to antibodies. Here, we present a protocol for the key aspects of the DNA-PAINT framework for both novice and expert users. This protocol describes the creation of DNA origami test samples, in situ sample preparation, multiplexed data acquisition, data simulation, super-resolution image reconstruction and post-processing such as drift correction, molecule counting (qPAINT) and particle averaging. Moreover, we provide an integrated software package, named Picasso, for the computational steps involved. The protocol is designed to be modular, so that individual components can be chosen and implemented per requirements of a specific application. The procedure can be completed in 1–2 d.

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Acknowledgements

We thank B. Rieger, S.S. Agasti, S. Strauss, D. Haas, J.B. Woehrstein and E. Woehrstein for helpful discussions. This work was supported by the German Research Foundation (DFG) through an Emmy Noether Fellowship (DFG JU 2957/1-1), the European Research Council (ERC) through an ERC Starting Grant (MolMap, grant agreement no. 680241), the Max Planck Society, the Max Planck Foundation and the Center for Nanoscience (CeNS). M.T.S. acknowledges support from the International Max Planck Research School for Molecular and Cellular Life Sciences (IMPRS-LS). T.S. acknowledges support from the DFG through the Graduate School of Quantitative Biosciences Munich (QBM). F.S. acknowledges support from the DFG through the SFB 1032 (Nanoagents for the spatiotemporal control of molecular and cellular reactions).

Author information

Author notes

    • Joerg Schnitzbauer
    •  & Maximilian T Strauss

    These authors contributed equally to this work.

Affiliations

  1. Department of Physics and Center for Nanoscience, Ludwig Maximilian University, Munich, Germany.

    • Joerg Schnitzbauer
    • , Maximilian T Strauss
    • , Thomas Schlichthaerle
    • , Florian Schueder
    •  & Ralf Jungmann
  2. Max Planck Institute of Biochemistry, Martinsried, Germany.

    • Joerg Schnitzbauer
    • , Maximilian T Strauss
    • , Thomas Schlichthaerle
    • , Florian Schueder
    •  & Ralf Jungmann

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Contributions

J.S. and M.T.S. contributed equally to this work. J.S. designed and developed the Picasso software suite. M.T.S. developed 'Picasso: Design' and 'Simulate' and performed in vitro experiments. T.S. developed antibody labeling strategies and performed in situ experiments. F.S. performed ultra-resolution experiments. R.J. conceived the study and supervised the project. All authors contributed to the writing of the manuscript.

Competing interests

R.J. is a cofounder of Ultivue, a startup company with an interest in commercializing DNA-PAINT technology.

Corresponding author

Correspondence to Ralf Jungmann.

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https://doi.org/10.1038/nprot.2017.024

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