Immunofluorescence refers to the combination of an antibody and a compound that will fluoresce when illuminated by light of a specific wavelength. The duo is also referred to as a fluorescently labeled antibody. Such an antibody can be used to visually determine the location of a target antigen in biological samples, typically by microscopic observation.
The fluorescent compound that is attached to an antibody is able to absorb light of a certain wavelength, the particular wavelength being dependent on the molecular construction of the compound. The absorption of the light confers additional energy to the compound. The energy must be relieved. This is accomplished by the emission of light, at a higher wavelength (and so a different color) than the absorbed radiation. It is this release of radiant energy that is the under-pinning for immunofluorescence.
Immunofluorescence microscopy can revel much detail about the processes inside cells. In a light microscopic application of the technique, sections of sample are exposed to the fluorescently labeled antibody. The large wavelength of visible light, relative to other forms of illumination such as laser light, does not allow details to be revealed at the molecular level. Still, details of the trafficking of a protein from the site of its manufacture to the surface of a cell, for example, is possible, by the application of different antibodies. The antibodies can be labeled with the same fluorescent compound but are applied at different times. An example of the power of this type of approach is the information that has been obtained concerning the pathway that the yeast known as Saccharomyces cervisiae uses to shuttle proteins out of the cell.
Resolution of details to the molecular level has been made possible during the 1990s with the advent of the technique of confocal laser microscopy. This technique employs a laser to sequentially scan samples at selected depths through the sample. These so-called optical sections can be obtained using laser illumination at several different wavelengths simultaneously. Thus, the presence of different antibodies that are labeled with fluorescent compounds that fluoresce at the different wavelengths can produce an image of the location of two antigens in the same sample at the same time.
The use of immunofluorescent compounds in combination with confocal microscopy has allowed the fluorescent probing of samples which do not need to be chemically preserved (or "fixed") prior to examination. The thin sections of sample that are examined in light microscopy often require such chemical fixation. While the fixation regimens have been designed to avoid change of the sample's internal structure, especially the chemistry and three-dimensional structure of the site of the antigen to which the antibody will bind, the avoidance of any form of chemical modification is preferred.
There are a multitude of fluorescent compounds available. Collectively these compounds are referred to as fluorochromes. A well-known example in biological and microbiological studies is the green fluorescent protein. This molecule is ring-like in structure. It fluoresces green when exposed to light in the ultraviolet or blue wavelengths. Other compounds such as fluorescein, rhodamine, phycoerythrin, and Texas Red, fluoresce at different wavelength and can produce different colors.
Immunofluorescence can be accomplished in a one-step or two-step reaction. In the first option, the fluorescently labeled antibody directly binds to the target antigen molecule. In the second option the target antigen molecule binds a socalled secondary antibody. Then, other antigenic sites in the sample that might also bind the fluorescent antibody are "blocked" by the addition of a molecule that more globally binds to antigenic sites. The secondary antibody then can itself be the target to which the fluorescently labeled antibody binds.
The use of antibodies to antigen that are critical to disease processes in microorganisms allow immunofluorescence to act as a detection and screening tool in the monitoring of a variety of materials. Foe example, research to adapt immunofluorescence to food monitoring is an active field. In the present, immunofluorescence provides the means by which organisms can be sorted using the technique of flow cytometry. As individual bacteria , for example, pass by a detector, the presence of fluorescence will register and cause the bacterium to be shuttled to a special collection reservoir. Thus, bacteria with a certain surface factor can be separated from the other bacteria in the population that do not possess the factor
See also Fluorescent dyes; Microscopy