Overview

A fluorescence microscope uses fluorescent chromophores called fluorochromes, which can absorb energy from a light source and then emit this energy as visible light. Fluorochromes include naturally fluorescent substances (such as chlorophylls) and fluorescent stains that are added to the specimen to create contrast. Dyes such as Texas red and FITC are examples of fluorochromes. Other examples include the nucleic acid dyes 4’,6’-diamidino-2-phenylindole (DAPI), and acridine orange.

The microscope irradiates the sample with short wavelength excitation, such as ultraviolet or blue light. The chromophores absorb the excitation light and emit visible light of longer wavelengths. The excitation light is then filtered out (partly because ultraviolet light is harmful to the eyes) so that only visible light passes through the ocular lens, producing an image of the specimen in bright colors against a dark background.

Fluorescence microscopes can identify pathogens, find particular species within an environment, or find the locations of particular molecules and structures within a cell. Approaches have also been developed to distinguish living from dead cells based on whether they take up particular fluorochromes. Sometimes, multiple fluorochromes are used on the same specimen to show different structures or features.

One of the most important applications of fluorescence microscopy is immunofluorescence, which is used to identify certain microbes by observing whether antibodies bind to them. (Antibodies are protein molecules the immune system produces that attach to specific pathogens to kill or inhibit them.) This technique has two approaches: direct immunofluorescence assay (DFA) and indirect immunofluorescence assay (IFA). In DFA, specific antibodies (e.g., those that target the rabies virus) are stained with a fluorochrome. If the specimen contains the targeted pathogen, one can observe the antibodies binding to the pathogen under the fluorescent microscope. This is a primary antibody stain because the stained antibodies attach directly to the pathogen.

In IFA, secondary antibodies are stained with a fluorochrome rather than primary antibodies. Secondary antibodies do not attach directly to the organism but bind to primary antibodies. When the unstained primary antibodies bind to the pathogen, the fluorescent secondary antibodies can be observed binding to the primary antibodies. Thus, the secondary antibodies are attached indirectly to the pathogen. Since multiple secondary antibodies can often attach to a primary antibody, IFA increases the number of fluorescent antibodies attached to the specimen, making it easier to visualize its features.

This text is adapted from Openstax, Microbiology 2e, Section 2.4: Staining Microscopic Specimens.

Procedure

In immunofluorescence microscopy, fluorophore labeled antibodies emit fluorescence upon binding a specific target or an antigen.

Immunofluorescence microscopy uses light to excite electrons of the fluorophore to a higher energy state. As they return to the ground state, the electrons release a longer wavelength of light.

This emission or fluorescence allows visualization of specific cells within tissues or particular proteins within cells.

Immunofluorescence can be direct when the primary fluorophore-tagged antibody binds to the target protein. Or, it can be indirect, where secondary antibodies tagged with fluorophores bind a specific primary antibody attached to the protein of interest. The resulting fluorescence is stronger than that emitted by direct immunofluorescence.

Direct immunofluorescence is used to detect abnormal protein aggregation in tissues, while indirect immunofluorescence can detect the circulating antibodies in the serum during autoimmune disease diagnosis.