Skip to main content
SpringerLink
Log in

Field emission scanning electron microscopy of plant cells

  • Original Papers
  • Published:
Protoplasma Aims and scope Submit manuscript

Summary

Two basic specimen preparation protocols that allow field emission scanning electron microscope imaging of intracellular structures in a wide range of plants are described. Both protocols depend on freeze fracturing to reveal areas of interest and selective removal of cytosol. Removal of cytosol was achieved either by macerating fixed tissues in a dilute solution of osmium tetroxide after freeze fracturing or by permeabilizing the membranes in saponin before fixation and subsequent freeze fracturing. Images of a variety of intracellular structures including all the main organelles as well as cytoskeletal components are presented. The permeabilization protocol can be combined with immunogold labelling to identify specific components such as microtubules. High-resolution three-dimensional imaging was combined with immunogold labelling of microtubules and actin cables in cell-free systems. This approach should be especially valuable for the study of dynamic cellular processes (such as cytoplasmic streaming) in live cells when used in conjunction with modern fluorescence microscopical techniques.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

DMSO:

dimethylsulfoxide

FESEM:

field emission scanning electron microscope (-scopy)

MTSB:

microtubule-stabilizing buffer

PBS:

phosphate-buffered saline

SEM:

scanning electron microscope (-scopy)

TEM:

transmission electron microscope (-scopy)

References

  • Allen TD, Goldberg MW (1993) High-resolution SEM in cell biology. Trends Cell Biol 3: 205–208

    Google Scholar 

  • —, Bennion GR, Rutherford SA, Reipert S, Ramalho A, Kiseleva E, Goldberg MW (1997) Macromolecular substructure in nuclear pore complexes by in-lens field-emission scanning microscopy. Scanning 19: 403–410

    Google Scholar 

  • Barnes SH (1992) Ultrastructural imaging of freeze-fractured plant cells in the scanning electron microscope. Microsc Res Tech 22: 160–169

    Google Scholar 

  • —, Blackmore S (1984) Scanning electron microscopy of chloroplast ultrastructure. Micron Microsc Acta 15: 187–194

    Google Scholar 

  • Blackmore S, Barnes SH, Claugher D (1984) Scanning electron microscopy of cytoskeletal components inAucuba japonica leaves. J Ultrastruct Res 86: 215–219

    Google Scholar 

  • Bond P, Donkin M, Moate R (1997) The development and evaluation of freeze-fracture/cytoplasmic maceration for the SEM to investigate algal ultrastructure. Micron 28: 433–438

    Google Scholar 

  • Cavalier-Smith T (1974) Basal body and flagellar development during vegetative cell cycle and the sexual cycle ofChlamydomonas reinhardtii. J Cell Sci 16: 529–556

    Google Scholar 

  • Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263: 802–805

    Google Scholar 

  • Chen Y, Centonze VE, Verkhovsky A, Borisy GG (1995) Imaging of cytoskeletal elements by low-temperature high-resolution scanning electron microscopy. J Microsc 179: 67–76

    Google Scholar 

  • Dibbayawan T, Cox G (1998) Flagellar root apparatus ofChlamydomonas: an immunocytochemical study using confocal microscopy and FESEM. In: Calderón Benavides HA, Jóse Yacamán M (eds) Electron microscopy 1988: proceedings of the 14th International Congress on Electron Microscopy, Cancun, Mexico, vol IV. Institute of Physics Publishing, Bristol, pp 729–730

    Google Scholar 

  • Fowke L, Dibbayawan T, Schwartz O, Harper J, Overall R (1999) Combined immunofluorescence and FESEM study of plasma membrane-associated organelles in highly vacuolated suspensor cells of white spruce somatic embryos. Cell Biol Int 23 (in press)

  • Fukudome H, Tanaka K (1992) A method for simultaneously revealing both the cytoskeleton and membranous cell organelles for scanning electron microscopy and its application to rat tissues. J Electron Microsc 41: 375–363

    Google Scholar 

  • Gunning BES, Steer MW (1996) Plant cell biology: structure and function. Jones and Bartlett, Sudbury, Mass

    Google Scholar 

  • Haggis GH (1992) Sample preparation for electron microscopy of internal cell structure. Microsc Res Tech 22: 151–159

    Google Scholar 

  • —, Phipps-Todd (1977) Freeze fracture for scanning electron microscopy. J Microsc 111: 193–201

    Google Scholar 

  • Harris E (1989) TheChlamydomonas source book: a comprehensive guide to biology and laboratory use. Academic Press, San Diego

    Google Scholar 

  • Hepler PK, Gunning BES (1998) Confocal fluorescence microscopy of plant cells. Protoplasma 201: 121–157

    Google Scholar 

  • Hermann R, Müller M (1997) Limits in high-resolution scanning electron microscopy: natural surfaces? Scanning 19: 337–342

    Google Scholar 

  • Inoué T (1985) High resolution scanning electron microscopic cytology: specimen preparation and intracellular structures observed by scanning electron microscopy. In: Müller M, Becker RP, Boyde A, Wolosewick JJ (eds) Science of biological specimen preparation. SEM Inc, Chicago, pp 245–256

    Google Scholar 

  • Kachar B, Reese TS (1988) The mechanism of cytoplasmic streaming in characean algal cells: sliding of endoplasmic reticulum along acting filaments. J Cell Biol 106: 1545–1552

    Google Scholar 

  • Knebel W, Quader H, Schnepf E (1990) Mobile and immobile endoplasmic reticulum in onion bulb epidermis cells: short- and long-term observations with a confocal laser scanning microscope. Eur J Cell Biol 52: 328–340

    Google Scholar 

  • Koga H, Tsukiboshi T, Uematsu T (1992) Application of an osmiummaceration technique to observe plant-microbe interfaces of Italian ryegrass and crown rust fungi by scanning electron microscopy. Can J Bot 70: 438–442

    Google Scholar 

  • — — — (1993) The structure of the cell organelles of plant leaves revealed by high resolution scanning electron microscopy. Bull Natl Grassland Res Inst Nishinasuno Tochigi 47: 37–42

    Google Scholar 

  • Lea PJ, Hollenberg MJ, Temkin RJ, Khan PA (1992) Chemical extraction of the cytosol using osmium tetroxide for high resolution scanning electron microscopy. Microsc Res Tech 22: 185–193

    Google Scholar 

  • Lindroth M, Bell PB, Fredriksson B-A, Liu X-D (1992) Preservation and visualization of molecular structure in detergent-extracted whole mounts of cultured cells. Microsc Res Tech 22: 130–150

    Google Scholar 

  • McLean B, Juniper BE (1993) The arrangement of actin bundles and chloroplasts in the nodal regions of characean internodal cells. Eur J Phycol 28: 33–37

    Google Scholar 

  • Müller WH, Van Aelst A, Van der Krift TP, Boekhout T (1994) Scanning electron microscopy of the septal pore cap of the basidiomyceteSchizophyllum commune. Can J Microbiol 40: 879–883

    Google Scholar 

  • Nagata T, Nemoto Y, Hasezawa S (1992) Tobacco BY-2 cell line as the “HeLa” cell in the cell biology of higher plants. Int Rev Cytol 132: 1–30

    Google Scholar 

  • Pawley J (1997) The development of field-emission scanning electron microscopy for imaging biological surfaces. Scanning 19: 324–336

    Google Scholar 

  • —, Erlandsen SL (1989) The case for low voltage high resolution scanning electron microscopy of biological samples. Scanning Microsc Suppl 3: 163–178

    Google Scholar 

  • — —, Bemrick WJ (1990) High resolution immunogold localization of the Giardia cyst wall antigens using secondary and backscatter electron imaging. J Histochem Cytochem 38: 625–632

    Google Scholar 

  • Peacock J, van Rensburg L, van der Merwe CF, Kruger H (1998) A FE-SEM study on the tobacco leaf epidermis. S Afr J Bot 64: 361–3671

    Google Scholar 

  • Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, Cormier MJ (1992) Primary structure of theAequorea victoria green fluorescent protein. Gene 111: 229–233

    Google Scholar 

  • Reichelt S, Ensikat H-J, Barthlott W, Volkmann D (1995) Visualization of immunogold-labelled cytoskeletal proteins by scanning electron microscopy. Eur J Cell Biol 67: 89–93

    Google Scholar 

  • Ringo DL (1967) Flagellar motion and fine structure of the flagellar apparatus inChlamydomonas. J Cell Biol 33: 543–571

    Google Scholar 

  • Ris H (1997) High-resolution field-emission scanning electron microscopy of nuclear pore complex. Scanning 19: 368–375

    Google Scholar 

  • —, Malecki M (1993) High resolution field emission scanning electron microscope imaging of internal cell structure after extraction from sections: a new approach to correlative ultrastructural and immunocytochemical studies. J Struct Biol 111: 148–157

    Google Scholar 

  • Tanaka K, Fukudome H (1991) Three-dimensional organization of the Golgi complex observed by scanning electron microscopy. J Electron Microsc Tech 17: 15–23

    Google Scholar 

  • —, Naguro T (1981) High resolution scanning electron microscopy of cell organelles by a new specimen preparation method. Biomed Res 2 Suppl: 63–70

    Google Scholar 

  • Temkin RJ, So DYF, Lea PJ (1993) Advantages of digitonin extraction to reveal the intracellular structure of rat glomerular podocytes for high-resolution scanning electron microscopy. Microsc Res Tech 26: 260–271

    Google Scholar 

  • Vesk PA, Rayns DG, Vesk M (1994) Imaging of plant microtubules with high resolution scanning electron microscopy. Protoplasma 182: 71–74

    Google Scholar 

  • —, Vesk M, Gunning BES (1996) Field emission scanning electron microscopy of microtubule arrays in higher plant cells. Protoplasma 195: 168–182

    Google Scholar 

  • Walther P, Müller M (1997) Double-layer coating for field-emission cryo-scanning electron microscopy: present state and applications. Scanning 19: 343–348

    Google Scholar 

  • Williamson RE (1993) Organelle movements. Annu Rev Plant Physiol 44: 181–202

    Google Scholar 

  • Wright RL, Salisbury J, Jarvik JW (1985) A nucleus-basal body connector inChlamydomonas reinhardtii that may function in basal body localization or segregation. J Cell Biol 101: 1903–1912

    Google Scholar 

Download references

Author information

Author notes

  1. Teresa P. Dibbayawan

    Present address: Biological Sciences, University of Sydney, Sydney, New South Wales, Australia

  2. Peter A. Vesk

    Present address: Biological Sciences, Macquarie University, Eastwood, New South Wales, Australia

Authors and Affiliations

  1. Electron Microscope Unit, University of Sydney, Sydney, New South Wales

    Maret Vesk, Peter A. Vesk & Elizabeth A. Egan

  2. Australian Key Centre for Microscopy and Microanalysis, University of Sydney, F09, Madsen Building, 2006, Sydney, NSW, Australia

    Maret Vesk & Teresa P. Dibbayawan

Authors
  1. Maret Vesk
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Teresa P. Dibbayawan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Peter A. Vesk
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Elizabeth A. Egan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Maret Vesk.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Vesk, M., Dibbayawan, T.P., Vesk, P.A. et al. Field emission scanning electron microscopy of plant cells. Protoplasma 210, 138–155 (2000). https://doi.org/10.1007/BF01276854

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01276854

Keywords

Search

Navigation