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DNA origami–based standards for quantitative fluorescence microscopy

Abstract

Validating and testing a fluorescence microscope or a microscopy method requires defined samples that can be used as standards. DNA origami is a new tool that provides a framework to place defined numbers of small molecules such as fluorescent dyes or proteins in a programmed geometry with nanometer precision. The flexibility and versatility in the design of DNA origami microscopy standards makes them ideally suited for the broad variety of emerging super-resolution microscopy methods. As DNA origami structures are durable and portable, they can become a universally available specimen to check the everyday functionality of a microscope. The standards are immobilized on a glass slide, and they can be imaged without further preparation and can be stored for up to 6 months. We describe a detailed protocol for the design, production and use of DNA origami microscopy standards, and we introduce a DNA origami rectangle, bundles and a nanopillar as fluorescent nanoscopic rulers. The protocol provides procedures for the design and realization of fluorescent marks on DNA origami structures, their production and purification, quality control, handling, immobilization, measurement and data analysis. The procedure can be completed in 1–2 d.

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Figure 1: Calibration structures for super-resolution microscopy.
Figure 2: Brightness standards.
Figure 3: AFM imaging of DNA origami structures.
Figure 4: Flowchart of the procedure.
Figure 5: Distance dependence on Mg2+ concentration.
Figure 6: caDNAno-assisted modification of DNA origami structures.
Figure 7: Immobilization and labeling of DNA origami structures.
Figure 8: Quality control of DNA origami structures.
Figure 9: Distance measurement.
Figure 10: Principle of the localization sorting in 3D.
Figure 11: Different degrees of bending of RRO and NRO.
Figure 12: Anticipated results.

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Acknowledgements

We gratefully acknowledge STED measurements from F. Göttfert and SIM measurements from L. Schermelleh. We thank R. Jungmann for the caDNAno file for the NRO structure. This work was supported by a starting grant (SiMBA, ERC-2010-StG-20091118) of the European Research Council, the Biophotonics IV program of the Federal Ministry of Education and Research (BMBF, VDI) (13N11461) and the German Research Foundation (DFG Ti329/6-1).

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J.J.S. developed and troubleshot the technique; M.R. performed dSTORM measurements; C.F. developed and troubleshot the software and algorithms for data analysis; E.P. developed and troubleshot the AFM measurements; B.W. carried out confocal measurements; T.D. produced the scaffold strands and quality-checked the structures; P.T. conceived the study and supervised the projects. All authors contributed to the writing of the manuscript.

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Correspondence to Philip Tinnefeld.

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Technische Universität Braunschweig has filed German and US patent applications covering parts of the work described in this manuscript.

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Schmied, J., Raab, M., Forthmann, C. et al. DNA origami–based standards for quantitative fluorescence microscopy. Nat Protoc 9, 1367–1391 (2014). https://doi.org/10.1038/nprot.2014.079

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