Immunogold labelling
Encyclopedia
Immunogold labelling or Immunogold staining (IGS) is a staining technique used in electron microscopy. Colloidal gold
particles are most often attached to secondary antibodies which are in turn attached to primary antibodies
designed to bind a specific protein
or other cell
component. Gold is used for its high electron density
which increases electron
scatter to give high contrast 'dark spots'.
First used in 1971, immunogold labelling has been applied to both transmission electron microscopy
and scanning electron microscopy, as well as brightfield microscopy. The labelling technique can be adapted to distinguish multiple objects by using differently-sized gold particles.
Immunogold labelling can introduce artefacts, as the gold particles reside some distance from the labelled object and very thin sectioning is required during sample preparation.
antigens. It was first applied in transmission electron microscopy (TEM) and was especially useful in highlighting protein
s found in low densities, such as some cell surface antigens. As the resolution of scanning electron microscopy (SEM) increased, so too did the need for nanoparticle-sized labels such as immunogold. In 1975, Horisberger and coworkers successfully visualised gold nanoparticles with a diameter of less than 30 nm
and this soon became an established SEM technique.
. Various other stages of sample preparation may then take place.
The prepared sample is then incubated with a specific antibody designed to bind the molecule of interest. Next, a secondary antibody which has gold particles attached is added, and it binds to the primary antibody. Gold can also be attached to protein A
or protein G
instead of a secondary antibody, as these proteins bind mammalian IgG Fc regions in a non-specific way.
The electron-dense gold particle can now be seen under an electron microscope as a black dot, indirectly labelling the molecule of interest.
by using two different-sized gold particles. An extension of this method used three different sized gold particles to track the localisation of regulatory peptides. A more complex method of multi-site labelling involves labelling opposite sides of an antigenic site
separately, the immunogold particles attached to both sides can then be viewed simultaneously.
containing silver ion
s. Gold particles then act as a nucleation
site and silver is deposited onto the particle. An example of the application of silver-enhanced immunogold labelling (IGSS) was in the identification of the pathogen
Erwinia amylovora.
labelling strategy). The precise location of the targeted molecule can therefore not be accurately calculated. This problem has been partially overcome by decreasing the size of the gold nanoparticle. Gold particles can be created with a diameter of 1 nm
(or lower) but another limitation is then realised—at these sizes the gold label becomes hard to distinguish from tissue structure.
Thin sections are required for immunogold labelling and these can produce misleading images; a thin slice of a cell component may not give an accurate view of its three-dimensional structure. For example, a microtubule
may appear as a 'spike' depending on which plane the sectioning occurred. To overcome this limitation serial sections can be taken, which can then be compiled into a three-dimensional
image.
A further limitation is that antibodies and gold particles cannot penetrate the resin
used to embed samples for imaging. Thus, only accessible molecules can be targeted and visualised. Labelling prior to embedding the sample can reduce the negative impact of this limitation.
Colloidal gold
Colloidal gold is a suspension of sub-micrometre-sized particles of gold in a fluid — usually water. The liquid is usually either an intense red colour , or a dirty yellowish colour ....
particles are most often attached to secondary antibodies which are in turn attached to primary antibodies
Primary and secondary antibodies
Primary and secondary antibodies are two groups of antibodies based on whether they target a target of interest directly or target another antibody that, in turn, is bound to a target of interest.-Primary:...
designed to bind a specific protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
or other cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
component. Gold is used for its high electron density
Electron density
Electron density is the measure of the probability of an electron being present at a specific location.In molecules, regions of electron density are usually found around the atom, and its bonds...
which increases electron
Electron
The electron is a subatomic particle with a negative elementary electric charge. It has no known components or substructure; in other words, it is generally thought to be an elementary particle. An electron has a mass that is approximately 1/1836 that of the proton...
scatter to give high contrast 'dark spots'.
First used in 1971, immunogold labelling has been applied to both transmission electron microscopy
Transmission electron microscopy
Transmission electron microscopy is a microscopy technique whereby a beam of electrons is transmitted through an ultra thin specimen, interacting with the specimen as it passes through...
and scanning electron microscopy, as well as brightfield microscopy. The labelling technique can be adapted to distinguish multiple objects by using differently-sized gold particles.
Immunogold labelling can introduce artefacts, as the gold particles reside some distance from the labelled object and very thin sectioning is required during sample preparation.
History
Immunogold labelling was first used in 1971 by Faulk and Taylor to identify SalmonellaSalmonella
Salmonella is a genus of rod-shaped, Gram-negative, non-spore-forming, predominantly motile enterobacteria with diameters around 0.7 to 1.5 µm, lengths from 2 to 5 µm, and flagella which grade in all directions . They are chemoorganotrophs, obtaining their energy from oxidation and reduction...
antigens. It was first applied in transmission electron microscopy (TEM) and was especially useful in highlighting protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s found in low densities, such as some cell surface antigens. As the resolution of scanning electron microscopy (SEM) increased, so too did the need for nanoparticle-sized labels such as immunogold. In 1975, Horisberger and coworkers successfully visualised gold nanoparticles with a diameter of less than 30 nm
and this soon became an established SEM technique.
Technique
First, a thin section of the sample is cut, often using a microtomeMicrotome
A microtome is a sectioning instrument that allows for the cutting of extremely thin slices of material, known as sections. Microtomes are an important device in microscopy preparation, allowing for the preparation of samples for observation under transmitted light or electron radiation...
. Various other stages of sample preparation may then take place.
The prepared sample is then incubated with a specific antibody designed to bind the molecule of interest. Next, a secondary antibody which has gold particles attached is added, and it binds to the primary antibody. Gold can also be attached to protein A
Protein A
Protein A is a 40-60 kDa MSCRAMM surface protein originally found in the cell wall of the bacterium Staphylococcus aureus. It is encoded by the spa gene and its regulation is controlled by DNA topology, cellular osmolarity, and a two-component system called ArlS-ArlR. It has found use in...
or protein G
Protein G
Protein G is an immunoglobulin-binding protein expressed in group C and G Streptococcal bacteria much like Protein A but with differing specificities. It is a 65-kDa and a 58 kDa cell surface protein that has found application in purifying antibodies through its binding to the Fc region...
instead of a secondary antibody, as these proteins bind mammalian IgG Fc regions in a non-specific way.
The electron-dense gold particle can now be seen under an electron microscope as a black dot, indirectly labelling the molecule of interest.
Labelling multiple objects
Immunogold labelling can be used to visualise more than one target simultaneously. This can be achieved in electron microscopyElectron microscope
An electron microscope is a type of microscope that uses a beam of electrons to illuminate the specimen and produce a magnified image. Electron microscopes have a greater resolving power than a light-powered optical microscope, because electrons have wavelengths about 100,000 times shorter than...
by using two different-sized gold particles. An extension of this method used three different sized gold particles to track the localisation of regulatory peptides. A more complex method of multi-site labelling involves labelling opposite sides of an antigenic site
Antigen
An antigen is a foreign molecule that, when introduced into the body, triggers the production of an antibody by the immune system. The immune system will then kill or neutralize the antigen that is recognized as a foreign and potentially harmful invader. These invaders can be molecules such as...
separately, the immunogold particles attached to both sides can then be viewed simultaneously.
Uses in brightfield microscopy
Although immunogold labelling is typically used for transmission electron microscopy, when the gold is 'silver-enhanced' it can be seen using brightfield microscopy. The silver enhancement increases the particle size, also making scanning electron microscopy possible. In order to produce the silver-enhanced gold particles, colloidal gold particles are placed in an acidic enhancing solutionSolution
In chemistry, a solution is a homogeneous mixture composed of only one phase. In such a mixture, a solute is dissolved in another substance, known as a solvent. The solvent does the dissolving.- Types of solutions :...
containing silver ion
Ion
An ion is an atom or molecule in which the total number of electrons is not equal to the total number of protons, giving it a net positive or negative electrical charge. The name was given by physicist Michael Faraday for the substances that allow a current to pass between electrodes in a...
s. Gold particles then act as a nucleation
Nucleation
Nucleation is the extremely localized budding of a distinct thermodynamic phase. Some examples of phases that may form by way of nucleation in liquids are gaseous bubbles, crystals or glassy regions. Creation of liquid droplets in saturated vapor is also characterized by nucleation...
site and silver is deposited onto the particle. An example of the application of silver-enhanced immunogold labelling (IGSS) was in the identification of the pathogen
Pathogen
A pathogen gignomai "I give birth to") or infectious agent — colloquially, a germ — is a microbe or microorganism such as a virus, bacterium, prion, or fungus that causes disease in its animal or plant host...
Erwinia amylovora.
Limitations
An inherent limitation to the immunogold technique is that the gold particle is around 15-30 nm away from the site to which the primary antibody is bound (when using a primary and secondary antibodiesPrimary and secondary antibodies
Primary and secondary antibodies are two groups of antibodies based on whether they target a target of interest directly or target another antibody that, in turn, is bound to a target of interest.-Primary:...
labelling strategy). The precise location of the targeted molecule can therefore not be accurately calculated. This problem has been partially overcome by decreasing the size of the gold nanoparticle. Gold particles can be created with a diameter of 1 nm
Nanometre
A nanometre is a unit of length in the metric system, equal to one billionth of a metre. The name combines the SI prefix nano- with the parent unit name metre .The nanometre is often used to express dimensions on the atomic scale: the diameter...
(or lower) but another limitation is then realised—at these sizes the gold label becomes hard to distinguish from tissue structure.
Thin sections are required for immunogold labelling and these can produce misleading images; a thin slice of a cell component may not give an accurate view of its three-dimensional structure. For example, a microtubule
Microtubule
Microtubules are a component of the cytoskeleton. These rope-like polymers of tubulin can grow as long as 25 micrometers and are highly dynamic. The outer diameter of microtubule is about 25 nm. Microtubules are important for maintaining cell structure, providing platforms for intracellular...
may appear as a 'spike' depending on which plane the sectioning occurred. To overcome this limitation serial sections can be taken, which can then be compiled into a three-dimensional
Three-dimensional space
Three-dimensional space is a geometric 3-parameters model of the physical universe in which we live. These three dimensions are commonly called length, width, and depth , although any three directions can be chosen, provided that they do not lie in the same plane.In physics and mathematics, a...
image.
A further limitation is that antibodies and gold particles cannot penetrate the resin
Resin
Resin in the most specific use of the term is a hydrocarbon secretion of many plants, particularly coniferous trees. Resins are valued for their chemical properties and associated uses, such as the production of varnishes, adhesives, and food glazing agents; as an important source of raw materials...
used to embed samples for imaging. Thus, only accessible molecules can be targeted and visualised. Labelling prior to embedding the sample can reduce the negative impact of this limitation.