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 accelerated electrons as a source of illumination. As the wavelength of an electron can be up to 100,000 times shorter than that of visible light photons, electron microscopes have a hi ...

. This staining technique is an equivalent of the

indirect immunofluorescence Immunofluorescence is a technique used for light microscopy with a fluorescence microscope and is used primarily on microbiological samples. This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to spec ...

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 usually either wine-red coloured (for spherical particles less than 100 nm) or blue/purple (for larger spherical particl ...

particles are most often attached to secondary

antibodies An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of ...

which are in turn attached to

primary 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 ...

designed to bind a specific

antigen In immunology, an antigen (Ag) is a molecule or molecular structure or any foreign particulate matter or a pollen grain that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune respon ...

or other

cell Cell most often refers to: * Cell (biology), the functional basic unit of life Cell may also refer to: Locations * Monastic cell, a small room, hut, or cave in which a religious recluse lives, alternatively the small precursor of a monastery ...

component. Gold is used for its high

electron density In quantum chemistry, 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 va ...

which increases

electron The electron ( or ) is a subatomic particle with a negative one elementary electric charge. Electrons belong to the first generation of the lepton particle family, and are generally thought to be elementary particles because they have n ...

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 ...

and scanning electron microscopy, as well as

brightfield microscopy Bright-field microscopy (BF) is the simplest of all the optical microscopy illumination techniques. Sample illumination is transmitted (i.e., illuminated from below and observed from above) white light, and contrast in the sample is caused by att ...

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

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

. 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 residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, res ...

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''. Important in science, microtomes are used in microscopy, all ...

. 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 is a 42 kDa surface protein originally found in the cell wall of the bacteria ''Staphylococcus aureus''. It is encoded by the ''spa'' gene and its regulation is controlled by DNA topology, cellular osmolarity, and a two-component system ...

or protein G instead of a secondary antibody, as these proteins bind mammalian

IgG Immunoglobulin G (Ig G) is a type of antibody. Representing approximately 75% of serum antibodies in humans, IgG is the most common type of antibody found in blood circulation. IgG molecules are created and released by plasma B cells. Each IgG an ...

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 property allows antibodies to activate the immune s ...

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. Long chains of amino acids are called proteins. Chains of fewer than twenty amino acids are called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides. ...

. 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

. 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 In computer science, ACID ( atomicity, consistency, isolation, durability) is a set of properties of database transactions intended to guarantee data validity despite errors, power failures, and other mishaps. In the context of databases, a ...

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 * Solutio ...

containing silver ions. Gold particles then act as a

nucleation In thermodynamics, nucleation is the first step in the formation of either a new thermodynamic phase or structure via self-assembly or self-organization within a substance or mixture. Nucleation is typically defined to be the process that deter ...

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 ( el, πάθος, "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 g ...

Erwinia amylovora Fire blight, also written fireblight, is a contagious disease affecting apples, pears, and some other members of the family Rosaceae. It is a serious concern to apple and pear producers. Under optimal conditions, it can destroy an entire orchard ...

''.

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 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 nm and have an inner diameter between 1 ...

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 (also: 3D space, 3-space or, rarely, tri-dimensional space) is a geometric setting in which three values (called '' parameters'') are required to determine the position of an element (i.e., point). This is the inform ...

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

resin In polymer chemistry and materials science, resin is a solid or highly viscous substance of plant or synthetic origin that is typically convertible into polymers. Resins are usually mixtures of organic compounds. This article focuses on nat ...

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