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 firstname.lastname@example.org.
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Electron Microscope" Immunochemistry 8, 1081-1083.
- 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.
- Roth J, Bendayan M, Orci L (1980) "FITC-Protein A-Gold Complex
for Light and Electron Microscopic Immunocytochemistry" J.
Histochem. Cytochem. 28, 55-57.
- 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.
- 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.
- Scopsi L, Larsson LI, Bastholm L, Nielsen
MH (1986) "Silver
Enhanced Colloidal Gold Probes as Markers for Scanning Electron
Microscopy" Histochemistry 86, 35-41.
- 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.
- Albrecht RM, Hodges GM, (eds) (1988) "Biotechnology and
Bioapplications of Colloidal Gold" Scanning Microscopy International,
- 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.
- 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.
- Molday RS, Maher P (1980) "A Review of Cell Surface Markers
and Labelling Techniques for Scanning Electron Microscopy" Histochem.
J, 12, 273-315.