Electron microscopy reveals mitochondrial DNA in discrete foci

Immunogold labeling or immunogold staining (IGS) is a staining technique used in

electron microscopy An electron microscope is a microscope that uses a beam of electrons as a source of illumination. It uses electron optics that are analogous to the glass lenses of an optical light microscope to control the electron beam, for instance focusing i ...

. This staining technique is an equivalent of the

indirect immunofluorescence Immunofluorescence (IF) is a light microscopy-based technique that allows detection and localization of a wide variety of target biomolecules within a cell or tissue at a quantitative level. The technique utilizes the binding specificity of antib ...

technique for visible light.

Colloidal gold Colloidal gold is a sol or colloidal suspension of nanoparticles of gold in a fluid, usually water. The colloid is coloured usually either wine red (for spherical particles less than 100 nm) or blue-purple (for larger spherical partic ...

particles are most often attached to secondary

antibodies An antibody (Ab) or immunoglobulin (Ig) is a large, Y-shaped protein belonging to the immunoglobulin superfamily which is used by the immune system to identify and neutralize antigens such as bacteria and viruses, including those that caus ...

which are in turn attached to primary antibodies designed to bind a specific

antigen In immunology, an antigen (Ag) is a molecule, moiety, foreign particulate matter, or an allergen, such as pollen, that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune response. ...

or other

cell Cell most often refers to: * Cell (biology), the functional basic unit of life * Cellphone, a phone connected to a cellular network * Clandestine cell, a penetration-resistant form of a secret or outlawed organization * Electrochemical cell, a de ...

component. Gold is used for its high

electron density Electron density or electronic density is the measure of the probability of an electron being present at an infinitesimal element of space surrounding any given point. It is a scalar quantity depending upon three spatial variables and is typical ...

which increases

electron The electron (, or in nuclear reactions) is a subatomic particle with a negative one elementary charge, elementary electric charge. It is a fundamental particle that comprises the ordinary matter that makes up the universe, along with up qua ...

scatter to give high contrast 'dark spots'. First used in 1971, immunogold labeling has been applied to both

transmission electron microscopy Transmission electron microscopy (TEM) is a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image. The specimen is most often an ultrathin section less than 100 nm thick or a suspension on a g ...

and

scanning electron microscopy A scanning electron microscope (SEM) is a type of electron microscope that produces images of a sample by scanning the surface with a focused beam of electrons. The electrons interact with atoms in the sample, producing various signals that ...

, as well as brightfield microscopy. The labeling technique can be adapted to distinguish multiple objects by using differently-sized gold particles. Immunogold labeling can introduce artifacts, as the gold particles reside some distance from the labelled object and very thin sectioning is required during sample preparation.

History

Immunogold labeling was first used in 1971 by Faulk and Taylor to identify ''

Salmonella ''Salmonella'' is a genus of bacillus (shape), rod-shaped, (bacillus) Gram-negative bacteria of the family Enterobacteriaceae. The two known species of ''Salmonella'' are ''Salmonella enterica'' and ''Salmonella bongori''. ''S. enterica'' ...

antigens In immunology, an antigen (Ag) is a molecule, moiety, foreign particulate matter, or an allergen, such as pollen, that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune response. An ...

. It was first applied in transmission electron microscopy (TEM) and was especially useful in highlighting

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...

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

microtome A microtome (from the Greek ''mikros'', meaning "small", and ''temnein'', meaning "to cut") is a cutting tool used to produce extremely thin slices of material known as ''sections'', with the process being termed microsectioning. Important in sc ...

. 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 Protein G is an immunoglobulin-binding protein expressed in group C and G streptococcal bacteria much like protein A but with differing binding specificities. It is a ~60-kDA (65 kDA for strain G148 and 58 kDa for strain C40) cell surface pro ...

instead of a secondary antibody, as these proteins bind mammalian IgG

Fc region The fragment crystallizable region (Fc region) is the tail region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. This region allows antibodies to activate the immune sys ...

s in a non-specific way. The electron-dense gold particle can now be seen under an electron microscope as a black dot, indirectly labeling the molecule of interest.

Labeling multiple objects

Immunogold labeling can be used to visualize more than one target simultaneously. This can be achieved in

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 Peptides are short chains of amino acids linked by peptide bonds. A polypeptide is a longer, continuous, unbranched peptide chain. Polypeptides that have a molecular mass of 10,000 Dalton (unit), Da or more are called proteins. Chains of fewer t ...

. A more complex method of multi-site labeling involves labeling opposite sides of an antigenic site separately, the immunogold particles attached to both sides can then be viewed simultaneously.

Uses in brightfield microscopy

Although immunogold labeling 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 An acid is a molecule or ion capable of either donating a proton (i.e. hydrogen cation, H+), known as a Brønsted–Lowry acid, or forming a covalent bond with an electron pair, known as a Lewis acid. The first category of acids are the ...

enhancing

solution Solution may refer to: * Solution (chemistry), a mixture where one substance is dissolved in another * Solution (equation), in mathematics ** Numerical solution, in numerical analysis, approximate solutions within specified error bounds * Solu ...

containing silver

ion An ion () is an atom or molecule with a net electrical charge. The charge of an electron is considered to be negative by convention and this charge is equal and opposite to the charge of a proton, which is considered to be positive by convent ...

s. Gold particles then act as a

nucleation In thermodynamics, nucleation is the first step in the formation of either a new Phase (matter), thermodynamic phase or Crystal structure, structure via self-assembly or self-organization within a substance or mixture. Nucleation is typically def ...

site and silver is deposited onto the particle. An example of the application of silver-enhanced immunogold labeling (IGSS) was in the identification of the

pathogen In biology, a pathogen (, "suffering", "passion" and , "producer of"), in the oldest and broadest sense, is any organism or agent that can produce disease. A pathogen may also be referred to as an infectious agent, or simply a Germ theory of d ...

'' 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 antibodies Primary and secondary antibodies are two groups of antibodies that are classified based on whether they bind to ''antigens or proteins'' directly or target another (primary) antibody that, in turn, is bound to an ''antigen or protein''. Primary A ...

labeling strategy). The precise location of the targeted molecule can therefore not be accurately calculated. Gold particles can be created with a diameter of 1 nm (or lower) but another limitation is then realized—at these sizes the gold label becomes hard to distinguish from tissue structure. Thin sections are required for immunogold labeling 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 Microtubules are polymers of tubulin that form part of the cytoskeleton and provide structure and shape to eukaryotic cells. Microtubules can be as long as 50 micrometres, as wide as 23 to 27 nanometer, nm and have an inner diameter bet ...

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 In geometry, a three-dimensional space (3D space, 3-space or, rarely, tri-dimensional space) is a mathematical space in which three values (''coordinates'') are required to determine the position (geometry), position of a point (geometry), poi ...

image. A further limitation is that antibodies and gold particles cannot penetrate the

resin A resin is a solid or highly viscous liquid that can be converted into a polymer. Resins may be biological or synthetic in origin, but are typically harvested from plants. Resins are mixtures of organic compounds, predominantly terpenes. Commo ...

used to embed samples for imaging. Thus, only accessible molecules can be targeted and visualized. Labeling prior to embedding the sample can reduce the negative impact of this limitation.

References