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. KenneyEmail author
  • Mike HeilemannEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1624)


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 



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.


  1. 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):7807–7812CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Turkowyd B, Virant D, Endesfelder U (2016) From single molecules to life: microscopy at the nanoscale. Anal Bioanal Chem 408(25):6885–6911CrossRefPubMedPubMedCentralGoogle Scholar
  3. 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):14919–14930CrossRefPubMedGoogle Scholar
  4. 4.
    Heilemann M (2010) Fluorescence microscopy beyond the diffraction limit. J Biotechnol 149(4):243–251CrossRefPubMedGoogle Scholar
  5. 5.
    Betzig E, Patterson GH, Sougrat R et al (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313(5793):1642–1645CrossRefPubMedGoogle Scholar
  6. 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):155–157CrossRefPubMedGoogle Scholar
  7. 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–E4399CrossRefPubMedPubMedCentralGoogle Scholar
  8. 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):18911–18916CrossRefPubMedPubMedCentralGoogle Scholar
  9. 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–E1110CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Thompson RE, Larson DR, Webb WW (2002) Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82(5):2775–2783CrossRefPubMedPubMedCentralGoogle Scholar
  11. 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):6172–6176CrossRefPubMedGoogle Scholar
  12. 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):2004–2021CrossRefPubMedGoogle Scholar
  13. 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):2415–2420CrossRefPubMedPubMedCentralGoogle Scholar
  14. 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):8–13CrossRefPubMedPubMedCentralGoogle Scholar
  15. 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):30462–30466CrossRefPubMedPubMedCentralGoogle Scholar
  16. 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):243–249CrossRefPubMedGoogle Scholar
  17. 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):14005CrossRefGoogle Scholar
  18. 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):1297–1308CrossRefGoogle Scholar
  19. 19.
    Endesfelder U, Finan K, Holden SJ et al (2013) Multiscale spatial organization of RNA polymerase in Escherichia coli. Biophys J 105(1):172–181CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Burgert A, Letschert S, Doose S et al (2015) Artifacts in single-molecule localization microscopy. Histochem Cell Biol 144(2):123–131CrossRefPubMedGoogle Scholar
  21. 21.
    Endesfelder U, Heilemann M (2014) Art and artifacts in single-molecule localization microscopy: beyond attractive images. Nat Methods 11(3):235–238CrossRefPubMedGoogle Scholar
  22. 22.
    Edelstein A, Amodaj N, Hoover K et al (2010) Computer control of microscopes using microManager. Curr Protoc Mol Biol. Chapter 14: Unit14.20Google Scholar
  23. 23.
    Wolter S, Schuttpelz M, Tscherepanow M et al (2010) Real-time computation of subdiffraction-resolution fluorescence images. J Microsc 237(1):12–22CrossRefPubMedGoogle Scholar
  24. 24.
    Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682CrossRefPubMedGoogle Scholar
  25. 25.
    Malkusch S, Heilemann M (2016) Extracting quantitative information from single molecule super-resolution imaging data with LAMA – localization microscopy analyzer. Sci Rep 6:34486CrossRefPubMedPubMedCentralGoogle Scholar
  26. 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):1001–1010CrossRefPubMedGoogle Scholar
  27. 27.
    Zessin PJM, Krüger CL, Malkusch S et al (2013) A hydrophilic gel matrix for single-molecule super-resolution microscopy. Opt Nanoscopy 2(1):4CrossRefGoogle Scholar
  28. 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):112–121CrossRefPubMedGoogle Scholar
  29. 29.
    Legant WR, Shao L, Grimm JB et al (2016) High-density three-dimensional localization microscopy across large volumes. Nat Methods 13(4):359–365CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Klar TA, Hell SW (1999) Subdiffraction resolution in far-field fluorescence microscopy. Opt Lett 24(14):954–956CrossRefPubMedGoogle Scholar
  31. 31.
    Vicidomini G, Moneron G, Han KY et al (2011) Sharper low-power STED nanoscopy by time gating. Nat Methods 8(7):571–573CrossRefPubMedGoogle Scholar
  32. 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):377–381CrossRefPubMedPubMedCentralGoogle Scholar
  33. 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):629–638CrossRefPubMedGoogle Scholar
  34. 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 34:226–231Google Scholar
  35. 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):14271–14276CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Loschberger A, Niehorster T, Sauer M (2014) Click chemistry for the conservation of cellular structures and fluorescent proteins: ClickOx. Biotechnol J 9(5):693–697CrossRefPubMedGoogle Scholar
  37. 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):21097–21102CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Tokunaga M, Imamoto N, Sakata-Sogawa K (2008) Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat Methods 5(2):159–161CrossRefPubMedGoogle Scholar

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
    Email author
  • Mike Heilemann
    • 1
    Email author
  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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