Immunogold Labelling in Scanning Electron Microscopy

The development of immunochemical techniques is one of the most rapidly emerging fields of study for biological and medical research. The recent parallel establishment of reliable immunological methods for producing good quality monoclonal and polyclonal antibodies, as of techniques for complexing proteins onto gold particles to form immunological probes, now allows researchers and medical workers to investigate a vast range of antigens in cells and tissues. Virtually every biological and microscopical laboratory in the world now has the ability to perform these investigations simply, effectively and safely through the availability of high quality protein-gold complexes.

Gold particles may be conjugated to primary antibodies for one step identification of antigens (eg. with anti Human IgG) but are more usually employed as secondary antibody/protein labels. The method of use is very similar to conventional systems employing other types of label, but is much more simple and has fewer of the disadvantages of other techniques. The gold conjugates are non-hazardous and can be used for a wide range of applications. The technique is now recognized as the method of choice for both transmission electron microscopy and, more recently, scanning electron microscopy.

Colloidal gold labelling techniques were first introduced by Faulk and Taylor (1971) when they absorbed anit-salmonella rabbit gamma globulins to gold particles for one step identification and localization of salmonella antigens. Indirect labelling techniques with gold probes was subsequently introduced by Romano et al (1974) who also reported gold labelling of Protein A for detection of primary immunoglobulins. The application to thin sections for electron microscopy was described in detail by Roth et al (1980) and since then the use of colloidal gold in transmission electron microscopy has grown at an enormous rate and has become virtually the only method worth considering for ultrastructural studies of cellular antigens. The major reason for this is the high electron density of the gold particles coupled with the ease with which different particle sizes can be used for examination at different magnifications. More recently, however, it has become clear that the strong emission of secondary electrons and backscattered electrons from gold particles make the gold probes ideal for study of surface antigens and macromolecules in the scanning electron microscope (de Harven et al, 1984).

For some time the identification of cellular components with immunogold labelling by SEM was restricted due to difficulties in resolving the particles and the charging of non-sputtered specimens under the electron beam (Hoyer et al 1979, de Harven et al 1984). Improved resolving power of SEM instruments, the use of backscattered electron imaging and deposition of cells directly onto metal stubs with carbon coating have greatly improved this situation and the method is now considered to be extremely sensitive and specific, giving rapid analysis of protein distribution over wide areas of cells and tissue structures. Numerous cells can be observed simultaneously allowing a ready interpretation of molecular patterns with the three dimensional structure.

In addition to electron imaging the small gold particles have also been visualized more readily in the SEM after silver enhancing (Scopsi et al, 1986). Some workers have also combined electron imaging of gold particles with X-ray emission using an energy dispersive analyzer in the SEM for immunolabelling studies (Eskelinen et al, 1988).

Gold probes are available in sizes ranging from 1-40nm for electron microscopy. While the resolving power of a scanning electron microscope is, with secondary electron imaging, better than 1nm, the possibility of ambiguity between small gold particles and tissue structures indicate that larger particle sizes are preferred and are best visualized by backscattered electron imaging. While all sizes of gold probe may be used to label tissue proteins, the sizes most commonly employed for SEM studies are 20-30nm.

These gold probes are available commercially as gold particles irreversibly bound to a range of secondary antibodies, Protein A, Protein G, and Streptavidin. The method of application to tissues has now become routine and is well documented (Albrecht et al, 1988). Specimens are prepared in much the same way as for normal SEM observation but with careful modifications in order to preserve antigenicity. Both direct (one step) and indirect (two step) methods of labelling are possible with SEM. Of these two, the indirect method is most frequently used, being flexible and very economical (Hodges et al, 1984). Multiple labelling of different cellular proteins on the same structures using different sizes of gold probes or gold probes in conjuction with other labels has also become a popular method in the SEM (Kay et al, 1978, Molday et al, 1980).

Immunogold labelling in the scanning electron microscope has, then, become established as a trusted technique and, with the commercial availability of high quality gold probes having consistent and reproducible performance, is now a routine method in the electron microscopy laboratory.

High quality BioSite immunogold reagents are available from ENERGY BEAM SCIENCES, INC., 29b Kripes Road East Granby, CT 06026-9669. Telephone toll free (800) 992-9037, or email ebs@ebsciences.com.

References

1. Faulk W, Taylor G (1979) "An Immunocolloid Method for the Electron Microscope" Immunochemistry 8, 1081-1083.
2. Romano E, Stolinsky C, Hugh-Jones N (1974) "Ultrastructural Localisation of Intracellular Antigens by the use of Protein A-Gold Complex" Immunochemistry 11, 521-522.
3. Roth J, Bendayan M, Orci L (1980) "FITC-Protein A-Gold Complex for Light and Electron Microscopic Immunocytochemistry" J. Histochem. Cytochem. 28, 55-57.
4. De Harven E, Leung R, Christensen H (1984) "A Novel Approach for Scanning Electron Microscopy of Colloidal Gold Labelled Cell Surfaces" J Cell Biol. 99, 53-57.
5. Hoyer LC, Lee JC, Bucana C. (1979) "Scanning Immunoelectron Microscopy for the Identification and Mapping of Two or More Antigens on Cell Surfaces" Scanning Electron Microscopy 1979, 3, 629-636.
6. Scopsi L, Larsson LI, Bastholm L, Nielsen MH (1986) "Silver Enhanced Colloidal Gold Probes as Markers for Scanning Electron Microscopy" Histochemistry 86, 35-41.
7. Eskelinen S, Peura R (1988) "Location and Identification of Colloidal Gold Particles on the Cell Surface with a Scanning Electron Microscope and Energy Dispersive Analyser" Scanning Electron Microscopy, 1988, 2, 1765-1774.
8. Albrecht RM, Hodges GM, (eds) (1988) "Biotechnology and Bioapplications of Colloidal Gold" Scanning Microscopy International, IL, USA.
9. Hodges GM, Smolira MA, Livingston DC (1984) "Scanning Electron Microscope Immunocytochemistry in Practice" Immunolabelling for Electron Microscopy, Eds Polak J. Varndell I, Elsevier Science Publishers BV, Amsterdam, 189-233.
10. Kay MM (1978) "Multiple Labelling Technique for Immuno-scanning Electron Microscopy" Principles and Techniques of Scanning Electron Microscopy, Vol. 6, Hayat MA (ed), Van Nostrand Reinhold, New York, 338-357.
11. Molday RS, Maher P (1980) "A Review of Cell Surface Markers and Labelling Techniques for Scanning Electron Microscopy" Histochem. J, 12, 273-315.