Skip to main content
SpringerLink
Log in

Environmental scanning electron microscopy (ESEM)—a versatile tool in studying plants

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

Abstract

Environmental scanning electron microscopy (ESEM) enables the investigation of hydrated and uncoated plant samples and the in situ observation of dynamic processes. Water vapor in the microscope chamber takes part in secondary electron detection and charge prevention. Two ESEM modes are available and offer a broad spectrum of applications. The environmental or wet mode prevents sample dehydration by the combination of sample cooling (5°C) and a vapor pressure of 4–6 Torr. In the low vacuum mode, the maximum chamber pressure is limited to 1 Torr (corresponding to about 5% relative humidity in the chamber) and allows the simultaneous use of a backscattered electron detector for imaging material contrast. A selection of characteristic plant samples and various applications are presented as a guide to ESEM for plant scientists. Leaf surfaces, trichomes, epicuticular waxes, and inorganic surface layers represent samples being comparatively resistant to dehydration, whereas callus cells and stigmatic tissue are examples for dehydration- and beam-sensitive samples. The potential of investigating dynamic processes in situ is demonstrated by studying anther opening, by tensile testing of leaves, and by performing hydration/dehydration experiments by changing the vapor pressure. Additionally, automated block-face imaging and serial sectioning using in situ ultramicrotomy is presented. The strengths and weaknesses of ESEM are discussed and it is shown that ESEM is a versatile tool in plant science.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  • Bache IC, Donald AM (1998) The structure of the gluten network in dough: a study using environmental scanning electron microscopy. J Cereal Sci 28:127–133

    Article  CAS  Google Scholar 

  • Bergmans L, Moisiadis P, Van Meerbeek B, Quirynen M, Lambrechts P (2005) Microscopic observation of bacteria: review highlighting the use of environmental SEM. Int Endod J 38:775–788

    Article  CAS  PubMed  Google Scholar 

  • Cheng YT, Rodak DE, Angelopoulos A, Gacek T (2005) Microscopic observations of condensation of water on lotus leaves. Appl Phys Lett 87:194112. doi:10.1063/1.2130392

    Article  Google Scholar 

  • Crang RFE, Klomparens KL (eds) (1988) Artifacts in biological electron microscopy. Plenum, New York

    Google Scholar 

  • Danilatos GD (1981) The examination of fresh or living plant material in an environmental scanning electron microscope. J Microsc 121:235–238

    Google Scholar 

  • Danilatos GD (1993) Introduction to the ESEM instrument. Microsc Res Tech 25:354–361

    Article  CAS  PubMed  Google Scholar 

  • Denk W, Horstmann H (2004) Serial block face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol 2(11):e329

    Article  PubMed  Google Scholar 

  • Donald AM (2003) The use of environmental scanning electron microscopy for imaging wet and insulating materials. Nat Mater 2:511–516

    Article  CAS  PubMed  Google Scholar 

  • Donald AM, Baker FS, Smith AC, Waldron KW (2003) Fracture of plant tissues and walls as visualized by environmental scanning electron microscopy. Ann Bot 92:73–77

    Article  CAS  PubMed  Google Scholar 

  • Fechner P, Wartewig S, Kiesow A, Heilmann A, Kleinebudde P, Neubert RHH (2005) Influence of water on molecular and morphological structure of various starches and starch derivatives. Starch 57:605–615

    Article  CAS  Google Scholar 

  • Fedel M, Caciagli P, Christé V, Caola I, Tessarolo F (2007) Microbial biofilm imaging ESEM vs HVSEM. GIT Imag Microsc 2:44–47

    Article  Google Scholar 

  • Hiscock SJ, Allen AM (2008) Diverse cell signaling pathways regulate pollen–stigma interactions: the search for consensus. New Phytol 179:286–317

    Article  CAS  PubMed  Google Scholar 

  • Iwano M, Entani T, Shiba H, Takayama S, Isogai A (2004) Calcium crystals in the anther of Petunia: the existence and biological significance in the pollination process. Plant Cell Physiol 45:40–47

    Article  CAS  PubMed  Google Scholar 

  • James B (2009) Advances in “wet” electron microscopy techniques and their application to the study of food structure. Trends Food Sci Tech 20:114–124

    Article  CAS  Google Scholar 

  • Kitching S, Donald AM (1998) Beam damage of propylene in the environmental scanning electron microscope: an FTIR study. J Microsc 190:357–365

    Article  CAS  Google Scholar 

  • Kirk SE, Skepper JN, Donald AM (2009) Application of environmental scanning electron microscopy to determine biological surface structure. J Microsc 233:205–224

    Article  CAS  PubMed  Google Scholar 

  • Kolb D, Müller M (2004) Light, conventional and environmental scanning electron microscopy of the trichomes of Cucurbita pepo subsp. pepo var. styriaca and histochemistry of glandular secretory products. Ann Bot 94:515–526

    Article  PubMed  Google Scholar 

  • Lich B, Wall D, Knott G (2009) High-throughput 3D cellular imaging. In: Papst MA, Zellnig G (eds) MC2009, vol. 2: life sciences. Verlag der TU, Graz, p 285. doi:10.3217/978-3-85125-062-6-288

    Google Scholar 

  • Mathieu C (1996) Principles and applications of the variable pressure scanning electron microscope. Eur Microsc Anal 9(September):13–14

    Google Scholar 

  • Méndez-Vilas A, Jódar-Reyes AB, González-Martín ML (2009) Ultrasmall liquid droplets on solid surfaces: production, imaging, and relevance for current wetting research. Small 5:1366–1390

    Article  PubMed  Google Scholar 

  • Muscariello L, Rosso F, Marino G, Giordano A, Barbarisi M, Cafiero G, Barbarisi A (2005) A critical overview of ESEM applications in the biological field. J Cell Physiol 205:328–334

    Article  CAS  PubMed  Google Scholar 

  • Nase M, Zankel A, Langer B, Baumann HJ, Grellmann W, Poelt P (2008) Investigation of the peel behavior of polyethylene/polybutene-1 peel films using in situ peel tests with environmental scanning electron microscopy. Polymer 49:5458–5466

    Article  CAS  Google Scholar 

  • Pathan AK, Bond J, Gaskin RE (2008) Sample preparation for scanning electron microscopy of plant surfaces—horses for courses. Micron 39:1049–1061

    Article  CAS  PubMed  Google Scholar 

  • Rossi MP, Gogotsi Y (2004) Environmental SEM studies of nanofiber-liquid interactions. Microsc Anal 18:21–23

    Google Scholar 

  • Royall CP, Thiel BL, Donald AM (2001) Radiation damage of water in environmental scanning electron microscopy. J Microsc 204:185–195

    Article  CAS  PubMed  Google Scholar 

  • Stokes DJ (2003) Recent advances in electron imaging, image interpretation and applications: environmental scanning electron microscopy. Philos Trans R Soc Lond A 316:2771–2787

    Article  Google Scholar 

  • Tattini M, Matteini P, Saracini E, Traversi ML, Giordano C, Agati G (2007) Morphology and biochemistry of non-glandular trichomes in Cistus salvifolius L. leaves growing in extreme habitats of the Mediterranean basin. Plant Biol 9:411–419

    Article  CAS  PubMed  Google Scholar 

  • Teppner H, Stabentheiner E (2006) Inga feuillei (Mimosaceae–Ingeae): anther opening and polyad presentation. Phyton (Horn) 46:141–158

    Google Scholar 

  • Teppner H, Stabentheiner E (2007) Anther opening, polyad presentation, pollenkitt and pollen adhesive in four Calliandra species (Mimosaceae–Ingeae). Phyton (Horn) 47:291–320

    Google Scholar 

  • Thiel BL, Donald AM (1998) In situ mechanical testing of fully hydrated carrots (Daucus carota) in the environmental SEM. Ann Bot 82:727–733

    Article  Google Scholar 

  • Timp W, Matsudaira P (2008) Electron microscopy of hydrated samples. Methods Cell Biol 89:391–407

    Article  PubMed  Google Scholar 

  • Werker E (2000) Trichome diversity and development. Adv Bot Res 31:1–35

    Article  Google Scholar 

  • Zankel A, Poelt P, Ingolic E, Gahleitner M, Grein C (2005) The fracture behavior of polymers—in situ investigations in the ESEM. GIT Imag Microsc 1:16–18

    Google Scholar 

  • Zankel A, Poelt P, Gahleitner M, Ingolic E, Grein C (2007) Tensile tests of polymers at low temperatures in the environmental scanning electron microscope: an improved cooling platform. Scanning 29:261–269

    Article  CAS  PubMed  Google Scholar 

  • Zankel A, Kraus B, Pölt P, Schaffer M, Ingolic E (2009) Ultramicrotomy in the ESEM, a versatile method for materials and life sciences. J Microsc 233:140–148

    Article  CAS  PubMed  Google Scholar 

  • Zheng T, Waldron KW, Donald AT (2009) Investigation of viability of plant tissue in the environmental scanning electron microscopy. Planta 230:1105–1113

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Their work and support was very helpful and we want to thank Regina Willfurth for her help in the sample preparation, Alexandra Jammer for providing the callus samples, Günther Zellnig for providing the imbedded Pelargonium sample for in situ ultramicrotomy, and Helga Hammer for her excellent work in the greenhouse. The constructive criticism of the reviewers is greatly acknowledged.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Institute of Plant Sciences, University of Graz, Schubertstraße 51, 8010, Graz, Austria

    Edith Stabentheiner

  2. Institute for Electron Microscopy, Graz University of Technology and Center for Electron Microscopy, Steyrergasse 17, 8010, Graz, Austria

    Armin Zankel & Peter Pölt

Authors
  1. Edith Stabentheiner
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Armin Zankel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Peter Pölt
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Edith Stabentheiner.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Stabentheiner, E., Zankel, A. & Pölt, P. Environmental scanning electron microscopy (ESEM)—a versatile tool in studying plants. Protoplasma 246, 89–99 (2010). https://doi.org/10.1007/s00709-010-0155-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00709-010-0155-3

Keywords

Search

Navigation