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

  • Christoph Spahn
  • Mathilda Glaesmann
  • Yunfeng Gao
  • Yong Hwee Foo
  • Marko Lampe
  • Linda J. Kenney
  • Mike Heilemann
Part of the Methods in Molecular Biology book series (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.

Key words

Super-resolution microscopy Single-molecule imaging Bacterial nucleoid Protein quantification Bacterial regulatory proteins 

Notes

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.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Christoph Spahn
    • 1
  • Mathilda Glaesmann
    • 1
  • Yunfeng Gao
    • 2
  • Yong Hwee Foo
    • 2
  • Marko Lampe
    • 3
  • Linda J. Kenney
    • 2
    • 4
    • 5
  • Mike Heilemann
    • 1
  1. 1.Institute of Physical and Theoretical ChemistryGoethe-University FrankfurtFrankfurtGermany
  2. 2.Mechanobiology Institute, T-LabNational University of SingaporeSingaporeSingapore
  3. 3.Advanced Light Microscopy Facility, European Molecular Biology LaboratoryHeidelbergGermany
  4. 4.University of Illinois, ChicagoChicagoUSA
  5. 5.Jesse Brown VA Medical CenterChicagoUSA

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