Scanning Electron Microscopy of Cell Surface Morphology

Samantha Passey1, Stéphanie Pellegrin1, Harry Mellor1

1 Mammalian Cell Biology Laboratory, Department of Biochemistry, School of Medical Sciences, University of Bristol
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.17
DOI:  10.1002/0471143030.cb0417s37
Online Posting Date:  December, 2007
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Abstract

The surface of metazoan cells is a landscape not clearly visualized by light microscopy. Many cells elaborate protrusive structures such as microvilli, filopodia, lamellipodia, and surface ruffles that play important roles in the interaction between the cell and its environment. The high resolution of scanning electron microscopy makes it an ideal technique for studies of the cell surface; however, preservation of fine surface structure can be problematic. Here we highlight the critical factors in sample preparation to ensure optimal high‐resolution imaging of the surface of mammalian cells and tissues. Curr. Protoc. Cell Biol. 37:4.17.1‐4.17.13. © 2007 by John Wiley & Sons, Inc.

Keywords: filopodia; microvilli; lamellepodia; SEM; actin

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Lymphocytes for Scanning Electron Microscopy
  • Alternate Protocol 1: Alternative Drying Protocol—HMDS Replacement
  • Alternate Protocol 2: Preparation of Intestinal Epithelial Cells for SEM
  • Alternate Protocol 3: Preparation of Other Adherent Cell Types for SEM
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Lymphocytes for Scanning Electron Microscopy

  Materials
  • 5 M nitric acid
  • Methanol
  • Poly‐L‐lysine (0.1% w/v, Sigma‐Aldrich)
  • Phosphate‐buffered saline ( appendix 2A)
  • Cells of interest (this procedure has been used for SEM examination of primary human lymphocytes and also mouse and human lymphocyte cell lines)
  • RPMI 1640 medium (Sigma‐Aldrich)
  • 2.5% glutaraldehyde (see recipe)
  • Sodium phosphate buffer, pH 7.4 (see recipe)
  • Sodium phosphate buffer, pH 6.0 (see recipe)
  • 0.5% osmium tetroxide (see recipe)
  • 25%, 50%, 75%, 90%, and 100% ethanol
  • Liquid CO 2
  • High‐purity argon
  • 13‐mm round glass coverslips
  • Glass petri dish
  • 80°C oven
  • 12‐well tissue culture plates
  • 1.5‐ml microcentrifuge tube
  • Parafilm
  • Shaking incubator with variable speed
  • Benchtop microcentrifuge
  • Critical point drying apparatus (e.g., Tousimis Samdri; http://www.tousimis.com)
  • Aluminum sample stubs (12‐mm, Agar Scientific)
  • Adhesive sample mounting pads (Adhesive Carbon Tabs, 12‐mm, Agar Scientific)
  • Gold sputter coater
  • Vacuum
  • Dehumidified environment (e.g., sealed plastic container in the presence of silica gel)
  • Additional reagents and equipment for counting cells using a hemacytometer (unit 1.1)

Alternate Protocol 1: Alternative Drying Protocol—HMDS Replacement

  • Hexamethyldisilazane (HMDS, Sigma)
  • Filter paper
  • Desiccator

Alternate Protocol 2: Preparation of Intestinal Epithelial Cells for SEM

  • Caco‐2 cell line from ECACC or ATCC
  • Cell culture medium (see recipe)
  • 1% osmium tetroxide (see recipe)
  • Transwell polycarbonate membrane inserts (1.1‐cm2 surface area; 0.4‐µm pore; Corning, no. 3401) placed in a 12‐well tissue culture dish
  • Humidified 37°C, 5% CO 2 incubator
  • Voltohmmeter
  • Sharp blade
  • Additional reagents and equipment for trypsinization of cells (unit 1.1)
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Figures

Videos

Literature Cited

Literature Cited
   Abitorabi, M.A., Pachynski, R.K., Ferrando, R.E., Tidswell, M., and Erle, D.J. 1997. Presentation of integrins on leukocyte microvilli: A role for the extracellular domain in determining membrane localization. J. Cell Biol. 139:563‐571.
   Boyde, A. and Wood, C. 1969. Preparation of animal tissues for surface‐scanning electron microscopy. J. Microsc. 90:221‐249.
   Braet, F., De Zanger, R., and Wisse, E. 1997. Drying cells for SEM, AFM and TEM by hexamethyldisilazane: A study on hepatic endothelial cells. J. Microsc. 186:84‐87.
   Brown, M. J., Nijhara, R., Hallam, J. A., Gignac, M., Yamada, K. M., Erlandsen, S. L., Delon, J., Kruhlak, M., and Shaw, S. 2003. Chemokine stimulation of human peripheral blood T lymphocytes induces rapid dephosphorylation of ERM proteins, which facilitates loss of microvilli and polarization. Blood 102:3890‐3899.
   Brunk, U., Collins, V. P, and Arro, E. 1981. The fixation, dehydration, drying and coating of cultured cells for SEM. J. Microsc. 123:121‐131.
   Davey, E.J., Thyberg, J., Conrad, D.H., and Severinson, E. 1998. Regulation of Cell Morphology in B Lymphocytes by IL‐4: Evidence for Induced Cytoskeletal Changes. J. Immunol. 160:5366‐5373.
   Dean, P. and Kenny, B. 2004. Intestinal barrier dysfunction by enteropathogenic Escherichia coli is mediated by two effector molecules and a bacterial surface protein. Mol. Microbiol. 54:665‐675.
   Grasset, E., Pinto, M., Dussaulx, E., Zweibaum, A., and Desjeux, J.F. 1984. Epithelial properties of human colonic carcinoma cell line Caco‐2: Electrical parameters. Am. J. Physiol. 247:C260‐267.
   Newell, D.G. 1980. The white cell system. In Biomedical Research Applications of Scanning Electron Microscopy (G.M. Hodges and R.C. Hallowes, eds.) pp. 219‐305. Academic Press, London.
   Peterson, M.D. and Mooseker, M.S. 1992. Characterization of the enterocyte‐like brush border cytoskeleton of the C2BBe clones of the human intestinal cell line, Caco‐2. J. Cell Sci. 102:581‐600.
   Pinto, M., Robine‐Leon, S., Appay, M‐D., Kedinger, M., Triadou, N., Dussaulx, E., Lacroix, B., Simon‐Assmann, P., Haffen, K., Fogh, J., and Zweibaum, A. 1983. Enterocyte‐like differentiation and polarization of the human colon carcinoma cell line Caco‐2 in culture. Biol. Cell 47:323‐330.
   Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, N.Y.
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