Skip to main content

Imaging of Bacterial Chromosome Organization by 3D Super-Resolution Microscopy

  • Protocol
  • First Online:
The Bacterial Nucleoid

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

Abstract

The bacterial nucleoid is highly organized, yet it is dynamically remodeled by cellular processes such as transcription, replication, or segregation. Many principles of nucleoid organization have remained obscure due to the inability of conventional microscopy methods to retrieve structural information beyond the diffraction limit of light. Structured illumination microscopy has recently been shown to provide new levels of spatial details on bacterial chromosome organization by surpassing the diffraction limit. Its ease of use and fast 3D multicolor capabilities make it a method of choice for imaging fluorescently labeled specimens at the nanoscale. We describe a simple high-throughput method for imaging bacterial chromosomes using this technique.

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

Access this chapter

Institutional subscriptions

References

  1. Postow L, Hardy CD, Arsuaga J, Cozzarelli NR (2004) Topological domain structure of the Escherichia coli chromosome. Genes Dev 18:1766–1779

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Deng S, Stein RA, Higgins NP (2005) Organization of supercoil domains and their reorganization by transcription. Mol Microbiol 57:1511–1521

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Le TBK, Imakaev MV, Mirny LA, Laub MT (2013) High-resolution mapping of the spatial organization of a bacterial chromosome. Science 342:731–734

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Marbouty M et al (2015) Condensin- and replication-mediated bacterial chromosome folding and origin condensation revealed by hi-C and super-resolution imaging. Mol Cell 59:588–602

    Article  CAS  PubMed  Google Scholar 

  5. Wang X et al (2015) Condensin promotes the juxtaposition of DNA flanking its loading site in Bacillus subtilis. Genes Dev 29:1661–1675

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Espéli O, Boccard F (2006) Organization of the Escherichia coli chromosome into macrodomains and its possible functional implications. J Struct Biol 156:304–310

    Article  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Heilemann M et al (2008) Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew Chem Int Ed 47:6172–6176

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Le Gall A et al (2016) Bacterial partition complexes segregate within the volume of the nucleoid. Nat Commun 7:12107. doi:10.1038/ncomms12107

    Article  PubMed  PubMed Central  Google Scholar 

  13. Ball G et al (2015) SIMcheck: a toolbox for successful super-resolution structured illumination microscopy. Sci Rep 5:15915

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Lal A, Shan C, Xi P (2016) Structured illumination microscopy image reconstruction algorithm. IEEE J Sel Top Quantum Electron PP:1–1

    Google Scholar 

  15. P. Křížek, T. Lukeš, M. Ovesný, K. Fliegel, G. M. Hagen (2015). SIMToolbox: a MATLAB toolbox for structured illumination fluorescence microscopy. Bioinformatics 32:318–320

    Google Scholar 

  16. Müller M, Mönkemöller V, Hennig S, Hübner W, Huser T (2016) Open-source image reconstruction of super-resolution structured illumination microscopy data in ImageJ. Nat Commun 7:10980

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This research was supported by funding from the European Research Council under the 7th Framework Program (FP7/2010-2015, ERC grant agreement 260787) and from the Agence Nationale pour la Recherche for projects HiResBacs (ANR-15-CE11-0023) and IBM (ANR-14-CE19-0025-02).3D-SIM experiments were performed at Montpellier RIO imaging. We acknowledge support from France-BioImaging (FBI, ANR-10-INSB-04).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Marcelo Nollmann .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Le Gall, A., Cattoni, D.I., Nollmann, M. (2017). Imaging of Bacterial Chromosome Organization by 3D Super-Resolution Microscopy. 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_19

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7098-8_19

  • 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