Skip to main content
SpringerLink
Log in

Nanometer-Scale Molecular Mapping by Super-resolution Fluorescence Microscopy

  • Protocol
  • First Online:
Fluorescent Microscopy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2440))

Abstract

The structural organization of macromolecules and their association in assemblies and organelles is key to understand cellular function. Super-resolution fluorescence microscopy has expanded our toolbox for examining such nanometer-scale cellular structures, by enabling positional mapping of proteins in situ. Here, we detail the workflow to build nanometer-scale maps focusing on two complementary super-resolution modalities: structured illumination microscopy (SIM) and stochastic optical reconstruction microscopy (STORM).

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

6. References

  1. Stahl PL et al (2016) Visualization and analysis of gene expression in tissue sections by spatial transcriptomics. Science 353(6294):78–82

    CAS  PubMed  Google Scholar 

  2. Lundberg E, Borner GHH (2019) Spatial proteomics: a powerful discovery tool for cell biology. Nat Rev Mol Cell Biol 20(5):285–302

    CAS  PubMed  Google Scholar 

  3. Sydor AM et al (2015) Super-resolution microscopy: from single molecules to supramolecular assemblies. Trends Cell Biol 25(12):730–748

    CAS  PubMed  Google Scholar 

  4. Sigal YM, Zhou R, Zhuang X (2018) Visualizing and discovering cellular structures with super-resolution microscopy. Science 361(6405):880–887

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Sahl SJ, Hell SW, Jakobs S (2017) Fluorescence nanoscopy in cell biology. Nat Rev Mol Cell Biol 18(11):685–701

    CAS  PubMed  Google Scholar 

  6. Schur FK (2019) Toward high-resolution in situ structural biology with cryo-electron tomography and subtomogram averaging. Curr Opin Struct Biol 58:1–9

    CAS  PubMed  Google Scholar 

  7. Heintzmann R, Ficz G (2007) Breaking the resolution limit in light microscopy. Methods Cell Biol 81:561–580

    CAS  PubMed  Google Scholar 

  8. Gustafsson MG (2000) Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc 198(Pt 2):82–87

    CAS  PubMed  Google Scholar 

  9. Klar TA et al (2000) Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc Natl Acad Sci U S A 97(15):8206–8210

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Hofmann M et al (2005) Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. Proc Natl Acad Sci U S A 102(49):17565–17569

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Betzig E et al (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313(5793):1642–1645

    CAS  PubMed  Google Scholar 

  12. Hess ST, Girirajan TP, Mason MD (2006) Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J 91(11):4258–4272

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3(10):793–795

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Sharonov A, Hochstrasser RM (2006) Wide-field subdiffraction imaging by accumulated binding of diffusing probes. Proc Natl Acad Sci U S A 103(50):18911–18916

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Schnitzbauer J et al (2017) Super-resolution microscopy with DNA-PAINT. Nat Protoc 12(6):1198–1228

    CAS  PubMed  Google Scholar 

  16. Schermelleh L et al (2019) Super-resolution microscopy demystified. Nat Cell Biol 21(1):72–84

    CAS  PubMed  Google Scholar 

  17. Mockl L, Moerner WE (2020) Super-resolution microscopy with single molecules in biology and beyond-essentials, current trends, and future challenges. J Am Chem Soc 142(42):17828–17844

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Gustafsson MG et al (2008) Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophys J 94(12):4957–4970

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Mennella V et al (2012) Subdiffraction-resolution fluorescence microscopy reveals a domain of the centrosome critical for pericentriolar material organization. Nat Cell Biol 14(11):1159–1168

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Schermelleh L et al (2008) Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science 320(5881):1332–1336

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Bates M et al (2007) Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science 317(5845):1749–1753

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Huang B et al (2008) Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319(5864):810–813

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Nguyen QPH et al (2020) Comparative super-resolution mapping of basal feet reveals a modular but distinct architecture in primary and motile cilia. Dev Cell 55(2):209–223. e7

    CAS  PubMed  Google Scholar 

  24. Liu Z et al (2020) A quantitative super-resolution imaging toolbox for diagnosis of motile ciliopathies. Sci Transl Med 12(535):eaay0071

    CAS  PubMed  Google Scholar 

  25. Mennella V (2016) In: Stahl RBaP (ed) Structured illumination microscopy, in encyclopedia of cell biology. Elsevier, Amsterdam, Netherlands, pp 86–98

    Google Scholar 

  26. Wu Y, Shroff H (2018) Faster, sharper, and deeper: structured illumination microscopy for biological imaging. Nat Methods 15(12):1011–1019

    CAS  PubMed  Google Scholar 

  27. Loschberger Anna NY, Ralf N, Marie-Christine S, Ricardo B, Teresa K, Markus S, Ingo K (2021) Super-resolution imaging by dual iterative structured illumination microscopy. bioRxiv. https://doi.org/10.1101/2021.05.12.443720

  28. Huang B, Bates M, Zhuang X (2009) Super-resolution fluorescence microscopy. Annu Rev Biochem 78:993–1016

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Sahl SJ, Hell SW (2019) High-resolution 3D light microscopy with STED and RESOLFT. In: Bille JF (ed) High resolution imaging in microscopy and ophthalmology: new Frontiers in biomedical optics. Springer Nature, Cham (CH), pp 3–32

    Google Scholar 

  30. Wassie AT, Zhao Y, Boyden ES (2019) Expansion microscopy: principles and uses in biological research. Nat Methods 16(1):33–41

    CAS  PubMed  Google Scholar 

  31. Mennella V et al (2014) Amorphous no more: subdiffraction view of the pericentriolar material architecture. Trends Cell Biol 24(3):188–197

    CAS  PubMed  Google Scholar 

  32. Lawo S et al (2012) Subdiffraction imaging of centrosomes reveals higher-order organizational features of pericentriolar material. Nat Cell Biol 14(11):1148–1158

    CAS  PubMed  Google Scholar 

  33. Mennella V, Hanna R, Kim M (2015) Subdiffraction resolution microscopy methods for analyzing centrosomes organization. Methods Cell Biol 129:129–152

    CAS  PubMed  Google Scholar 

  34. Bowler M et al (2019) High-resolution characterization of centriole distal appendage morphology and dynamics by correlative STORM and electron microscopy. Nat Commun 10(1):993

    PubMed  PubMed Central  Google Scholar 

  35. Gao L et al (2014) 3D live fluorescence imaging of cellular dynamics using Bessel beam plane illumination microscopy. Nat Protoc 9(5):1083–1101

    CAS  PubMed  Google Scholar 

  36. York AG et al (2012) Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy. Nat Methods 9(7):749–754

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Sydor AM et al (2018) PPP1R35 is a novel centrosomal protein that regulates centriole length in concert with the microcephaly protein RTTN. elife 7

    Google Scholar 

  38. Los GV et al (2008) HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem Biol 3(6):373–382

    CAS  PubMed  Google Scholar 

  39. Keppler A et al (2003) A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat Biotechnol 21(1):86–89

    CAS  PubMed  Google Scholar 

  40. Gautier A et al (2008) An engineered protein tag for multiprotein labeling in living cells. Chem Biol 15(2):128–136

    CAS  PubMed  Google Scholar 

  41. Szymborska A et al (2013) Nuclear pore scaffold structure analyzed by super-resolution microscopy and particle averaging. Science 341(6146):655–658

    CAS  PubMed  Google Scholar 

  42. Schindelin J et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682

    Article  CAS  PubMed  Google Scholar 

  43. Winterflood CM et al (2015) Dual-color 3D superresolution microscopy by combined spectral-demixing and biplane imaging. Biophys J 109(1):3–6

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Testa I et al (2010) Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength. Biophys J 99(8):2686–2694

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Ries J (2020) SMAP: a modular super-resolution microscopy analysis platform for SMLM data. Nat Methods 17(9):870–872

    CAS  PubMed  Google Scholar 

  46. Sage D et al (2019) Super-resolution fight club: assessment of 2D and 3D single-molecule localization microscopy software. Nat Methods 16(5):387–395

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Liu Z et al (2020) Super-resolution microscopy and FIB-SEM imaging reveal parental centriole-derived, hybrid cilium in mammalian multiciliated cells. Dev Cell 55(2):224–236. e6

    PubMed  PubMed Central  Google Scholar 

  48. Gingras AC, Abe KT, Raught B (2019) Getting to know the neighborhood: using proximity-dependent biotinylation to characterize protein complexes and map organelles. Curr Opin Chem Biol 48:44–54

    CAS  PubMed  Google Scholar 

  49. May DG, Roux KJ (2019) BioID: a method to generate a history of protein associations. Methods Mol Biol 2008:83–95

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Reiter JF, Leroux MR (2017) Genes and molecular pathways underpinning ciliopathies. Nat Rev Mol Cell Biol 18(9):533–547

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Shi X et al (2017) Super-resolution microscopy reveals that disruption of ciliary transition-zone architecture causes Joubert syndrome. Nat Cell Biol 19(10):1178–1188

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Kner P et al (2009) Super-resolution video microscopy of live cells by structured illumination. Nat Methods 6(5):339–342

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Shao L et al (2011) Super-resolution 3D microscopy of live whole cells using structured illumination. Nat Methods 8(12):1044–1046

    CAS  PubMed  Google Scholar 

  54. Demmerle J et al (2017) Strategic and practical guidelines for successful structured illumination microscopy. Nat Protoc 12(5):988–1010

    CAS  PubMed  Google Scholar 

  55. Ball G et al (2012) A cell biologist's guide to high resolution imaging. Methods Enzymol 504:29–55

    CAS  PubMed  Google Scholar 

  56. Wallace W, Schaefer LH, Swedlow JR (2001) A workingperson's guide to deconvolution in light microscopy. BioTechniques 31(5):1076–1078. 1080, 1082 passim

    CAS  PubMed  Google Scholar 

  57. Righolt CH et al (2014) Three-dimensional structured illumination microscopy using Lukosz bound apodization reduces pixel negativity at no resolution cost. Opt Express 22(9):11215–11227

    PubMed  Google Scholar 

  58. Righolt CH et al (2013) Image filtering in structured illumination microscopy using the Lukosz bound. Opt Express 21(21):24431–24451

    PubMed  Google Scholar 

  59. Thompson MA et al (2010) Molecules and methods for super-resolution imaging. Methods Enzymol 475:27–59

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This work was funded by the UK Medical Research Council intramural project MC_UU_00025/13 to Vito Mennella. We are grateful to Jonathan Shewring at Zeiss, inc. for help with data acquisition and reconstruction with Elyra 7 and SIM2 algorithm.

Author information

Authors and Affiliations

  1. MRC Toxicology Unit, School of Biological Sciences, University of Cambridge, Cambridge, UK

    Vito Mennella

  2. Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong SAR, China

    Zhen Liu

Authors
  1. Vito Mennella
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhen Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Vito Mennella .

Editor information

Editors and Affiliations

  1. Department of Microbiology and Immunology, Center for Human Immunology, The University of Western Ontario, London, ON, Canada

    Bryan Heit

Rights and permissions

Reprints and permissions

Copyright information

© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Mennella, V., Liu, Z. (2022). Nanometer-Scale Molecular Mapping by Super-resolution Fluorescence Microscopy. In: Heit, B. (eds) Fluorescent Microscopy. Methods in Molecular Biology, vol 2440. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2051-9_18

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2051-9_18

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2050-2

  • Online ISBN: 978-1-0716-2051-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation