Skip to main content
SpringerLink
Log in

Sequential Super-Resolution Imaging of Bacterial Regulatory Proteins, the Nucleoid and the Cell Membrane in Single, Fixed E. coli Cells

  • Protocol
  • First Online:
The Bacterial Nucleoid

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1624))

Abstract

Despite their small size and the lack of compartmentalization, bacteria exhibit a striking degree of cellular organization, both in time and space. During the last decade, a group of new microscopy techniques emerged, termed super-resolution microscopy or nanoscopy, which facilitate visualizing the organization of proteins in bacteria at the nanoscale. Single-molecule localization microscopy (SMLM) is especially well suited to reveal a wide range of new information regarding protein organization, interaction, and dynamics in single bacterial cells. Recent developments in click chemistry facilitate the visualization of bacterial chromatin with a resolution of ~20 nm, providing valuable information about the ultrastructure of bacterial nucleoids, especially at short generation times. In this chapter, we describe a simple-to-realize protocol that allows determining precise structural information of bacterial nucleoids in fixed cells, using direct stochastic optical reconstruction microscopy (dSTORM). In combination with quantitative photoactivated localization microscopy (PALM), the spatial relationship of proteins with the bacterial chromosome can be studied. The position of a protein of interest with respect to the nucleoids and the cell cylinder can be visualized by super-resolving the membrane using point accumulation for imaging in nanoscale topography (PAINT). The combination of the different SMLM techniques in a sequential workflow maximizes the information that can be extracted from single cells, while maintaining optimal imaging conditions for each technique.

The original version of this chapter was revised. An erratum to this chapter can be found at DOI 10.1007/978-1-4939-7098-8_25

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

Access this chapter

Log in via an institution

Institutional subscriptions

References

  1. Rojas E, Theriot JA, Huang KC (2014) Response of Escherichia coli growth rate to osmotic shock. Proc Natl Acad Sci U S A 111(21):7807–7812

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Turkowyd B, Virant D, Endesfelder U (2016) From single molecules to life: microscopy at the nanoscale. Anal Bioanal Chem 408(25):6885–6911

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Furstenberg A, Heilemann M (2013) Single-molecule localization microscopy-near-molecular spatial resolution in light microscopy with photoswitchable fluorophores. Phys Chem Chem Phys 15(36):14919–14930

    Article  PubMed  Google Scholar 

  4. Heilemann M (2010) Fluorescence microscopy beyond the diffraction limit. J Biotechnol 149(4):243–251

    Article  CAS  PubMed  Google Scholar 

  5. Betzig E, Patterson GH, Sougrat R et al (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313(5793):1642–1645

    Article  CAS  PubMed  Google Scholar 

  6. Manley S, Gillette JM, Patterson GH et al (2008) High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nat Methods 5(2):155–157

    Article  CAS  PubMed  Google Scholar 

  7. Stracy M, Lesterlin C, Garza de Leon F et al (2015) Live-cell superresolution microscopy reveals the organization of RNA polymerase in the bacterial nucleoid. Proc Natl Acad Sci U S A 112(32):E4390–E4399

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Lew MD, Lee SF, Ptacin JL et al (2011) Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus. Proc Natl Acad Sci U S A 108(46):E1102–E1110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Thompson RE, Larson DR, Webb WW (2002) Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82(5):2775–2783

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Heilemann M, van de Linde S, Schuttpelz M et al (2008) Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew Chem Int Ed Engl 47(33):6172–6176

    Article  CAS  PubMed  Google Scholar 

  12. Kolb HC, Finn MG, Sharpless KB (2001) Click chemistry: diverse chemical function from a few good reactions. Angew Chem Int Ed Engl 40(11):2004–2021

    Article  CAS  PubMed  Google Scholar 

  13. Salic A, Mitchison TJ (2008) A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc Natl Acad Sci U S A 105(7):2415–2420

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Ferullo DJ, Cooper DL, Moore HR et al (2009) Cell cycle synchronization of Escherichia coli using the stringent response, with fluorescence labeling assays for DNA content and replication. Methods 48(1):8–13

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Raulf A, Spahn CK, Zessin PJM et al (2014) Click chemistry facilitates direct labelling and super-resolution imaging of nucleic acids and proteins. RSC Adv 4(57):30462–30466

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Spahn C, Endesfelder U, Heilemann M (2014) Super-resolution imaging of Escherichia coli nucleoids reveals highly structured and asymmetric segregation during fast growth. J Struct Biol 185(3):243–249

    Article  CAS  PubMed  Google Scholar 

  17. Spahn C, Cella-Zannacchi F, Endesfelder U et al (2015) Correlative super-resolution imaging of RNA polymerase distribution and dynamics, bacterial membrane and chromosomal structure in Escherichia coli. Methods Appl Fluoresc 3(1):14005

    Article  Google Scholar 

  18. Foo YH, Spahn C, Zhang H et al (2015) Single cell super-resolution imaging of E. coli OmpR during environmental stress. Integr Biol (Camb) 7(10):1297–1308

    Article  CAS  Google Scholar 

  19. Endesfelder U, Finan K, Holden SJ et al (2013) Multiscale spatial organization of RNA polymerase in Escherichia coli. Biophys J 105(1):172–181

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Burgert A, Letschert S, Doose S et al (2015) Artifacts in single-molecule localization microscopy. Histochem Cell Biol 144(2):123–131

    Article  CAS  PubMed  Google Scholar 

  21. Endesfelder U, Heilemann M (2014) Art and artifacts in single-molecule localization microscopy: beyond attractive images. Nat Methods 11(3):235–238

    Article  CAS  PubMed  Google Scholar 

  22. Edelstein A, Amodaj N, Hoover K et al (2010) Computer control of microscopes using microManager. Curr Protoc Mol Biol. Chapter 14: Unit14.20

    Google Scholar 

  23. Wolter S, Schuttpelz M, Tscherepanow M et al (2010) Real-time computation of subdiffraction-resolution fluorescence images. J Microsc 237(1):12–22

    Article  CAS  PubMed  Google Scholar 

  24. Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682

    Article  CAS  PubMed  Google Scholar 

  25. Malkusch S, Heilemann M (2016) Extracting quantitative information from single molecule super-resolution imaging data with LAMA – localization microscopy analyzer. Sci Rep 6:34486

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Michelsen O, de Mattos T, Joost M, Jensen PR et al (2003) Precise determinations of C and D periods by flow cytometry in Escherichia coli K-12 and B/r. Microbiology 149(Pt 4):1001–1010

    Article  CAS  PubMed  Google Scholar 

  27. Zessin PJM, Krüger CL, Malkusch S et al (2013) A hydrophilic gel matrix for single-molecule super-resolution microscopy. Opt Nanoscopy 2(1):4

    Article  Google Scholar 

  28. Qu D, Wang G, Wang Z et al (2011) 5-Ethynyl-2′-deoxycytidine as a new agent for DNA labeling: detection of proliferating cells. Anal Biochem 417(1):112–121

    Article  CAS  PubMed  Google Scholar 

  29. Legant WR, Shao L, Grimm JB et al (2016) High-density three-dimensional localization microscopy across large volumes. Nat Methods 13(4):359–365

    Article  PubMed  PubMed Central  Google Scholar 

  30. Klar TA, Hell SW (1999) Subdiffraction resolution in far-field fluorescence microscopy. Opt Lett 24(14):954–956

    Article  CAS  PubMed  Google Scholar 

  31. Vicidomini G, Moneron G, Han KY et al (2011) Sharper low-power STED nanoscopy by time gating. Nat Methods 8(7):571–573

    Article  CAS  PubMed  Google Scholar 

  32. Mortensen KI, Churchman LS, Spudich JA et al (2010) Optimized localization analysis for single-molecule tracking and super-resolution microscopy. Nat Methods 7(5):377–381

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Endesfelder U, Malkusch S, Fricke F et al (2014) A simple method to estimate the average localization precision of a single-molecule localization microscopy experiment. Histochem Cell Biol 141(6):629–638

    Article  CAS  PubMed  Google Scholar 

  34. Ester M, Kriegel H-P, Sander J et al (1996) A density-based algorithm for discovering clusters in a density-based algorithm for discovering clusters in large spatial databases with noise. Data Min Knowl Discov Databases 34:226–231

    Google Scholar 

  35. Hein B, Willig KI, Hell SW (2008) Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell. Proc Natl Acad Sci U S A 105(38):14271–14276

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Loschberger A, Niehorster T, Sauer M (2014) Click chemistry for the conservation of cellular structures and fluorescent proteins: ClickOx. Biotechnol J 9(5):693–697

    Article  PubMed  Google Scholar 

  37. Subach FV, Malashkevich VN, Zencheck WD et al (2009) Photoactivation mechanism of PAmCherry based on crystal structures of the protein in the dark and fluorescent states. Proc Natl Acad Sci U S A 106(50):21097–21102

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Tokunaga M, Imamoto N, Sakata-Sogawa K (2008) Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat Methods 5(2):159–161

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

C.S., M.G., and M.H. acknowledge funding by the German Science Foundation (DFG, grant CEF 115). The authors are grateful to Luke Lavis for kindly providing the Hoechst-JF646 dye. LJK is supported by VA IBX-000372 and NIH AIR21-123640 grants and an RCE in Mechanobiology from the Ministry of Education, Singapore.

Author information

Authors and Affiliations

  1. Institute of Physical and Theoretical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 7, 60438, Frankfurt, Germany

    Christoph Spahn, Mathilda Glaesmann & Mike Heilemann

  2. Mechanobiology Institute, T-Lab, National University of Singapore, 5A Engineering Drive 1, Singapore, 117411, Singapore

    Yunfeng Gao, Yong Hwee Foo & Linda J. Kenney

  3. Advanced Light Microscopy Facility, European Molecular Biology Laboratory, Otto-Meyerhofstr. 1, 69117, Heidelberg, Germany

    Marko Lampe

  4. University of Illinois, Chicago, Chicago, IL, 60612, USA

    Linda J. Kenney

  5. Jesse Brown VA Medical Center, Chicago, IL, 60612, USA

    Linda J. Kenney

Authors
  1. Christoph Spahn
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mathilda Glaesmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yunfeng Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yong Hwee Foo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Marko Lampe
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Linda J. Kenney
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Mike Heilemann
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Linda J. Kenney or Mike Heilemann .

Editor information

Editors and Affiliations

  1. UMR CNRS 7241, INSERM U1050, CIRB, Collège de France, Paris, France

    Olivier Espéli

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Spahn, C. et al. (2017). Sequential Super-Resolution Imaging of Bacterial Regulatory Proteins, the Nucleoid and the Cell Membrane in Single, Fixed E. coli Cells. In: Espéli, O. (eds) The Bacterial Nucleoid. Methods in Molecular Biology, vol 1624. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7098-8_20

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7098-8_20

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7097-1

  • Online ISBN: 978-1-4939-7098-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation