In Situ Super-Resolution Imaging of Genomic DNA with OligoSTORM and OligoDNA-PAINT

Part of the Methods in Molecular Biology book series (MIMB, volume 1663)


OligoSTORM and OligoDNA-PAINT meld the Oligopaint technology for fluorescent in situ hybridization (FISH) with, respectively, Stochastic Optical Reconstruction Microscopy (STORM) and DNA-based Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) to enable in situ single-molecule super-resolution imaging of nucleic acids. Both strategies enable ≤20 nm resolution and are appropriate for imaging nanoscale features of the genomes of a wide range of species, including human, mouse, and fruit fly (Drosophila).

Key words

Single-molecule Super-resolution Genome Chromosomes Chromatin FISH Oligopaint STORM DNA-PAINT OligoSTORM OligoDNA-PAINT 



The authors would like to thank members of the Wu, Zhuang, and Yin laboratories for extensive conversations and experience. This work was supported by grants from the NIH to the laboratories of C.-t W. (GM085169, DP1GM106412, RM1HG008525), X.Z. (R01GM105637), and P.Y. (1R01EB018659, 1-U01-MH106011), X.Z. is a Howard Hughes Medical Institute investigator. In addition, B.J.B. and A.N.B. were supported by Damon Runyon Postdoctoral Fellowships.


  1. 1.
    Huang B, Babcock H, Zhuang X (2010) Breaking the diffraction barrier: Super-resolution imaging of cells. Cell 143:1047–1058CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Sengupta P, Van Engelenburg S, Lippincott-Schwartz J (2012) Visualizing cell structure and function with point-localization superresolution imaging. Dev Cell 23:1092–1102CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Thompson MA, Lew MD, Moerner WE (2012) Extending microscopic resolution with single-molecule imaging and active control. Ann Rev Biophys 41:321–342CrossRefGoogle Scholar
  4. 4.
    Godin AG, Lounis B, Cognet L (2014) Super-resolution microscopy approaches for live cell imaging. Biophys J 107:1777–1784CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Beliveau BJ, Boettiger AN, Avendaño MS, Jungmann R, McCole RB, Joyce EF, Kim-Kiselak C, Bantignies F, Fonseka CY, Erceg J, Hannan MA, Hoang HG, Colognori D, Lee JT, Shih WM, Yin P, Zhuang X, CT W (2015) Single-molecule super-resolution imaging of chromosomes and in situ haplotype visualization using Oligopaint FISH probes. Nat Commun 6:7147. doi: 10.1038/ncomms8147 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Boettiger AN, Bintu B, Moffitt JR, Wang S, Beliveau BJ, Fudenberg G, Imakaev M, Mirny LA, C-t W, Zhuang X (2016) Super-resolution imaging reveals distinct chromatin folding for different epigenetic states. Nature 529:418–422CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Beliveau BJ, Joyce EF, Apostolopoulos N, Yilmaz F, Fonseka CY, McCole RB, Chang Y, Li JB, Senaratne TN, Williams BR, Rouillard JM, CT W (2012) Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes. Proc Natl Sci U S A 109:21301–21306CrossRefGoogle Scholar
  8. 8.
    Beliveau BJ, Apostolopoulos NA, Wu CT (2014) Visualizing genomes with Oligopaint FISH probes. Curr Protoc Mol Biol 105:Unit 14.23PubMedGoogle Scholar
  9. 9.
    Rust M, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3:793–796CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Huang B, Wang W, Bates M, Zhuang X (2008) Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319:810–813CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Bates M, Huang B, Dempsey G, Zhuang X (2007) Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science 317:1749–1753CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Sharanov A, Hochstrasser RM (2006) Wide-field subdiffraction imaging by accumulated binding of diffusing probes. Proc Natl Acad Sci U S A 103:18911–18916CrossRefGoogle Scholar
  13. 13.
    Jungmann R, Steinhauer C, Scheible M, Kuzyk A, Tinnefeld P, Simmel FC (2010) Single-molecule kinetics and super-resolution microscopy by fluorescence imaging of transient binding on DNA origami. Nano Lett 10:4756–4761CrossRefPubMedGoogle Scholar
  14. 14.
    Jungmann R, Avendaño MS, Woehrstein JB, Dai M, Shih WM, Yin P (2014) Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT. Nat Methods 11:313–318CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Bates M, Jones SA, Zhuang X (2010) Stochastic optical reconstruction microscopy (STORM) – a method for superresolution fluorescence imaging. In: Yuste R (ed) Imaging: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  16. 16.
    Dai M, Jungmann R, Yin P (2016) Optical imaging of individual biomolecules in densely packed clusters. Nat Nanotechnol 11:798–807CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Jungmann R, Avendaño MS, Dai M, Woehrstein JB, Agasti SS, Feiger Z, Rodal A, Yin P (2016) 2016 Quantitative super-resolution imaging with qPAINT. Nat Methods 13:439–442CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Chen KH, Boettiger AN, Moffitt JR, Wang S, Zhuang X (2015) Spatially resolved, highly multiplexed RNA profiling in single cells. Science 348:aaa6090CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Wang S, J-H S, Beliveau BJ, Bintu B, Moffitt JR, CT W, Zhuang X (2016) Spatial organization of chromatin domains and compartments in single chromosomes. Science 353:598–602CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Bates M, Blosser TR, Zhuang X (2005) Short-range spectroscopic ruler based on a single-molecule optical switch. Phys Rev Lett 94:108101CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Dempsey GT, Vaughan JC, Chen KH, Bates M, Zhuang X (2011) Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging. Nat Methods 8:1027–1036CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Murgha Y, Beliveau Bm Semrau K, Schwartz D, Wu CT, Gulari E, Rouillard JM (2015) Combined in vitro transcription and reverse transcription to amplify and label complex synthetic oligonucleotide probe libraries. BioTechniques 58:301–307CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Moffit JR, Zhuang X (2016) RNA imaging with multiplexed error-robust fluorescence in situ hybridization. Methods Enzymol 572:1–49CrossRefGoogle Scholar
  24. 24.
    Schmidt TL, Beliveau BJ, Uca YO, Theilmann M, Da Cruz F, CT W, Shih WM (2015) Scalable amplification of strand subsets from chip-synthesized oligonucleotide libraries. Nat Commun 6:8634. doi: 10.1038/ncomms9634 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Olivier N, Keller D, Gönczy P, Manley S (2013) Resolution doubling in 3D-STORM imaging through improved buffers. PLoS One 8:e69004CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Aitken EC, Marshall RA, Puglisi JD (2008) An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophys J 94:826–183CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of GeneticsHarvard Medical SchoolBostonUSA
  2. 2.Wyss Institute for Biologically Inspired EngineeringHarvard UniversityBostonUSA
  3. 3.Department of Systems BiologyHarvard Medical SchoolBostonUSA
  4. 4.Howard Hughes Medical InstituteCambridgeUSA
  5. 5.Department of Chemistry and Chemical BiologyHarvard UniversityCambridgeUSA
  6. 6.Department of Developmental BiologyStanford UniversityStanfordUSA
  7. 7.Department of PhysicsHarvard UniversityCambridgeUSA

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