Advertisement

Environmental Scanning Electron Microscopy in Cell Biology

  • J. E. McGregor
  • L. T. L. Staniewicz
  • S. E. Guthrie (neé Kirk)
  • A. M. Donald
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 931)

Abstract

Environmental scanning electron microscopy (ESEM) (1) is an imaging technique which allows hydrated, insulating samples to be imaged under an electron beam. The resolution afforded by this technique is higher than conventional optical microscopy but lower than conventional scanning electron microscopy (CSEM). The major advantage of the technique is the minimal sample preparation needed, making ESEM quick to use and the images less susceptible to the artifacts that the extensive sample preparation usually required for CSEM may introduce. Careful manipulation of both the humidity in the microscope chamber and the beam energy are nevertheless essential to prevent dehydration and beam damage artifacts. In some circumstances it is possible to image live cells in the ESEM (2).

In the following sections we introduce the fundamental principles of ESEM imaging before presenting imaging protocols for plant epidermis, mammalian cells, and bacteria. In the first two cases samples are imaged using the secondary electron (topographic) signal, whereas a transmission technique is employed to image bacteria.

Key words

Bacteria Hydrated STEM ESEM Plant epidermis Stomatal pores Mammalian cells Native state Surface imaging 

Notes

Acknowledgements

The authors wish to thank T. W. Fairhead, D. J. Stokes, D. Waller, J. N. Skepper, A. Grant, Z. Wang, C. ffrench-Constant, A.A. R.Webb, P. J. Franks, and A. Luzhynskaya for their involvement with the development of these techniques.

References

  1. 1.
    Danilatos GD (1993) Introduction to the ESEM instrument. Microsc Res Tech 25:354–361PubMedCrossRefGoogle Scholar
  2. 2.
    Zheng T, Waldron KW, Donald AM (2009) Investigation of viability of plant tissue in the environmental scanning electron microscopy. Planta 230:1105–1113PubMedCrossRefGoogle Scholar
  3. 3.
    Goldstein JI, Newbury DE, Echlin P, Joy DC, Fiori C, Lifshin E (1981) Scanning electron microscopy and X-ray microanalysis. Plenum Press, New YorkCrossRefGoogle Scholar
  4. 4.
    Donald AM (2003) The use of environmental scanning electron microscopy for imaging wet and insulating materials. Nat Mater 2:511–516PubMedCrossRefGoogle Scholar
  5. 5.
    Stokes DJ (2008) Principles and practice of variable pressure/environmental scanning electron microscopy (VP-ESEM). Wiley, Chichester, UKCrossRefGoogle Scholar
  6. 6.
    Haynes WM (2011) (ed) CRC handbook of chemistry and physics, CRC Press, Boca Raton, FloridaGoogle Scholar
  7. 7.
    Tai SSW, Tang XM (2001) Manipulating biological samples for environmental scanning electron microscopy observation. Scanning 23:267–272PubMedCrossRefGoogle Scholar
  8. 8.
    Cameron RE, Donald AM (1994) Minimizing sample evaporation in the environmental scanning electron-microscope. J Microsc 173:227–237CrossRefGoogle Scholar
  9. 9.
    Philips Electron Optics (1996) Environmental scanning electron microscopy: an introduction to ESEM. Robert Johnson Associates, El Dorado Hills, CaliforniaGoogle Scholar
  10. 10.
    Toth M, Baker FS (2004) Secondary electron imaging at gas pressures in excess of 15 torr. Microsc Microanal 10(suppl2):1062–1063Google Scholar
  11. 11.
    Jackson JD (1999) Classical electrodynamics, 3rd edn. Wiley, New YorkGoogle Scholar
  12. 12.
    Thomson NM, Channon K, Mokhtar NA, Staniewicz L, Rai R, Roy I, Sato S, Tsuge T, Donald AM, Summers D, Sivaniah E (2011) Imaging internal features of whole, unfixed bacteria. Scanning 33:59–68PubMedCrossRefGoogle Scholar
  13. 13.
    McGregor JE, Donald AM (2010) ESEM imaging of dynamic biological processes: the closure of stomatal pores. J Microsc 239:135–141PubMedGoogle Scholar
  14. 14.
    Kirk SE, Skepper J, Donald AM (2009) Application of environmental scanning electron microscopy to determine biological surface structure. J Microsc 233:205–224PubMedCrossRefGoogle Scholar
  15. 15.
    Collins SC, Pope RK, Scheetz RW, Ray RI, Wagner PA, Little BJ (1993) Advantages of environmental scanning electron microscopy in studies of microorganisms. Microsc Res Tech 25(398):405Google Scholar
  16. 16.
    Bogner A, Jouneau PH, Thollet G, Basset D, Gauthier C (2007) A history of scanning electron microscopy developments: towards “wet-STEM” imaging. Micron 38:390–401PubMedCrossRefGoogle Scholar
  17. 17.
    Stabentheiner E, Zankel A, Polt P (2010) Environmental scanning electron microscopy (ESEM)—a versatile tool in studying plants. Protoplasma 246:89–99PubMedCrossRefGoogle Scholar
  18. 18.
    Royall CP, Donald AM (2002) Optimisation of the environmental scanning electron microscope for observation of drying of matt water-based lacquers. Scanning 24:305–313PubMedCrossRefGoogle Scholar
  19. 19.
    Royall CP, Thiel BL, Donald AM (2001) Radiation damage of water in environmental scanning electron microscopy. J Microsc 204:185–195PubMedCrossRefGoogle Scholar
  20. 20.
    Guthrie S (2008) Exploration of the use of ESEM for the study of biological materials. PhD thesis, University of CambridgeGoogle Scholar
  21. 21.
    McGregor JE (2010) Imaging dynamic biological processes. PhD thesis, University of CambridgeGoogle Scholar
  22. 22.
    Mestres P, Putz N, Laue M (2003) Applications of ESEM to the study of biomedical specimens. Microsc Microanal 9(suppl3):490–491Google Scholar
  23. 23.
    McGregor JE, Wang Z, ffrench-Constant C, Donald AM (2010) Microscopy of myelination. In: Mendez-Vilas A, Diaz Alvarez J (eds) Microscopy: science, technology, applications and education, 2nd edn. Formatex Research Center, Badajoz, Spain, pp 1185–1195Google Scholar
  24. 24.
    Stokes DJ, Rea SM, Best SM, Bonfield W (2003) Electron microscopy of mammalian cells in the absence of fixing, freezing, dehydration, or specimen coating. Scanning 25:181–184PubMedCrossRefGoogle Scholar
  25. 25.
    Staniewicz L, Donald AM, Stokes DJ, Thompson N, Sivaniah E, Grant A, Bulmer D, Anjam Khan CM (2011) The application of STEM and in-situ controlled dehydration to bacterial systems using ESEM, Scanning. doi: 10.1002/sca.21000Google Scholar
  26. 26.
    Graumann PL (2007) Cytoskeletal elements in bacteria. Annu Rev Microbiol 61:589–618PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • J. E. McGregor
    • 1
    • 2
  • L. T. L. Staniewicz
    • 2
    • 3
  • S. E. Guthrie (neé Kirk)
    • 2
  • A. M. Donald
    • 2
  1. 1.The School of Biological SciencesUniversity of BristolBristolUK
  2. 2.The Cavendish Laboratory, Department of PhysicsUniversity of CambridgeCambridgeUK
  3. 3.The Department of Materials Science and MetallurgyCambridgeUK

Personalised recommendations