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Optical imaging of individual biomolecules in densely packed clusters

Abstract

Recent advances in fluorescence super-resolution microscopy have allowed subcellular features and synthetic nanostructures down to 10–20 nm in size to be imaged. However, the direct optical observation of individual molecular targets (5 nm) in a densely packed biomolecular cluster remains a challenge. Here, we show that such discrete molecular imaging is possible using DNA-PAINT (points accumulation for imaging in nanoscale topography)—a super-resolution fluorescence microscopy technique that exploits programmable transient oligonucleotide hybridization—on synthetic DNA nanostructures. We examined the effects of a high photon count, high blinking statistics and an appropriate blinking duty cycle on imaging quality, and developed a software-based drift correction method that achieves <1 nm residual drift (root mean squared) over hours. This allowed us to image a densely packed triangular lattice pattern with 5 nm point-to-point distance and to analyse the DNA origami structural offset with ångström-level precision (2 Å) from single-molecule studies. By combining the approach with multiplexed exchange-PAINT imaging, we further demonstrated an optical nanodisplay with 5 × 5 nm pixel size and three distinct colours with <1 nm cross-channel registration accuracy.

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Figure 1: Principle and requirements of DMI.
Figure 2: Systematic characterization of the blinking requirements and optimization of the DNA-PAINT imaging quality.
Figure 3: Principle and performance of DNA nanostructure templated drift correction.
Figure 4: Systematic quality analysis of 5 nm grid super-resolution image.
Figure 5: Discrete molecular imaging of 5 nm grid structure.
Figure 6: Discrete molecular imaging with complex patterns and multiplexed visualization.

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Acknowledgements

We thank M. Avendaño, J. Wöhrstein, F. Schüder, G. Church, W. Shih, D. Zhang, S. Agasti, E. Winfree and P. Rothemund for helpful discussions, and C. Myhrvold, N. Liu, W. Sun, X. Chen, F. Vigneault and H. Tang for helpful comments on the manuscript. This work is supported by a National Institutes of Health (NIH) Director's New Innovator Award (1DP2OD007292), an NIH Transformative Research Award (1R01EB018659), an NIH grant (5R21HD072481), an Office of Naval Research (ONR) Young Investigator Program Award (N000141110914), ONR grants (N000141010827 and N000141310593), a National Science Foundation (NSF) Faculty Early Career Development Award (CCF1054898), an NSF grant (CCF1162459) and a Wyss Institute for Biologically Engineering Faculty Startup Fund to P.Y. M.D. acknowledges support from the HHMI International Predoctoral Fellowship. R.J. acknowledges support from the Alexander von Humboldt-Foundation through a Feodor-Lynen Fellowship.

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Contributions

M.D. conceived of and designed the study, designed and performed the experiments, developed the software, analysed the data and wrote the manuscript. R.J. conceived of and designed the study, interpreted the data and critiqued the manuscript. P.Y. conceived of, designed and supervised the study, interpreted the data and wrote the manuscript. All authors reviewed and approved the manuscript.

Corresponding author

Correspondence to Peng Yin.

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Competing interests

The authors have filed a patent application. P.Y. and R.J. are co-founders of Ultivue, Inc., a start-up company with interests in commercializing the reported technology.

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Dai, M., Jungmann, R. & Yin, P. Optical imaging of individual biomolecules in densely packed clusters. Nature Nanotech 11, 798–807 (2016). https://doi.org/10.1038/nnano.2016.95

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