Super-resolution labelling with Action-PAINT

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Recent advances in localization-based super-resolution microscopy have enabled researchers to visualize single molecular features down to individual molecular components (~5 nm), but do not yet allow manipulation of single-molecule targets in a user-prescribed, context-dependent manner. Here we report an ‘Action-PAINT’ (PAINT, point accumulation for imaging in nanoscale topography) strategy for super-resolution labelling upon visualization on single molecules. This approach monitors and localizes DNA binding events in real time with DNA-PAINT, and upon visualization of binding to a desired location, photo-crosslinks the DNA to affix the molecular label. We showed the efficiency of 3-cyanovinylcarbazole nucleoside photo-inducible crosslinking on single molecular targets and developed a software package for real-time super-resolution imaging and crosslinking control. We then benchmarked our super-resolution labelling method on synthetic DNA nanostructures and demonstrated targeted multipoint labelling on various complex patterns with 30 nm selectivity. Finally, we performed targeted in situ labelling on fixed microtubule samples with a 40 nm target size and custom-controlled, subdiffraction spacing.

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Fig. 1: Principle of super-resolution single-molecule labelling.
Fig. 2: Crosslinking efficiency test and targeted single-molecule labelling.
Fig. 3: Multipoint super-resolution patterning.
Fig. 4: In situ super-resolution labelling on microtubules.

Data availability

Datasets generated during the study are available from the corresponding authors upon request.

Code availability

Custom computer programs used during the study are available from the corresponding authors upon request.

Change history

  • 01 October 2019

    In this Article originally published, the ORCID number for the author Mingjie Dai was missing; it should have been 0000-0002-8665-4966. This has now been corrected in all versions of the Article.


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The authors thank R. Barish, H. Soundarajan, S. Agasti, J. Woehrstein and R. Jungmann for preliminary work on aspects of earlier versions of the project and for helpful discussions, and W. Shih, H. Sasaki, B. Beliveau, E. Boyden and J. Paulsson for helpful discussions. This work was supported by a National Institutes of Health (NIH) Director’s New Innovator Award (1DP2OD007292), an NIH Transformative Research Award (1R01EB018659), an NIH Pioneer Award (1DP1GM133052), an NIH grant (5R21HD072481), an Office of Naval Research (ONR) Young Investigator Program Award (N000141110914), ONR grants (N000141010827, N000141310593 and N000141812549), a National Science Foundation (NSF) Faculty Early Career Development Award (CCF1054898), an NSF grant (CCF1162459) and the Molecular Robotics Initiative fund at the Wyss Institute for Biologically Engineering Faculty to P.Y. M.D. acknowledges support from a HHMI International Predoctoral Fellowship, Department Fellowship from Systems Biology Department at the Harvard Medical School, and a Technology Development Fellowship from the Wyss Institute at Harvard University. S.K.S. acknowledges support from a Human Frontier Science Program (HFSP) fellowship (LT000048/2016-L).

Author information

M.D. and P.Y. conceived and designed the study, N.L. and M.D. designed and performed the experiments and analysed the data, M.D. developed the software, S.K.S. provided advice and assistance with the microtubule sample preparation. P.Y. supervised the study. All the authors wrote and approved the manuscript.

Correspondence to Mingjie Dai or Peng Yin.

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Competing interests

A US patent (US App No. 15/104,570) has been filed that covers the concepts reported in this work (inventors, R. Barish and P.Y.). P.Y. is cofounder of Ultivue Inc. and NuProbe Global.

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Supplementary Information

Supplementary Materials 1, Supplementary Materials 2, Supplementary Methods 3, Supplementary Notes 4, Supplementary Figs. 1–15 and 5 and Supplementary Tables 1–4.

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