thumb|278x278px|Vasculature of porcine [[skin under fluorescence (Smooth muscle actin with AlexaFluor 488). Green = smooth muscle actin (SMA) with Alexa 488 fluorophore. Blue = DAPI counterstain. Red = auto-fluorescence. ]]

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 antibodies and antigens. The specific region an antibody recognizes on an antigen is called an epitope. Several antibodies can recognize the same epitope but differ in their binding affinity. The antibody with the higher affinity for a specific epitope will surpass antibodies with a lower affinity for the same epitope.

By conjugating the antibody to a fluorophore, the position of the target biomolecule is visualized by exciting the fluorophore and measuring the emission of light in a specific predefined wavelength using a fluorescence microscope. It is imperative that the binding of the fluorophore to the antibody itself does not interfere with the immunological specificity of the antibody or the binding capacity of its antigen.

Immunofluorescence is a widely used example of immunostaining (using antibodies to stain proteins) and is a specific example of immunohistochemistry (the use of the antibody-antigen relationship in tissues). This technique primarily utilizes fluorophores to visualize the location of the antibodies, while others provoke a color change in the environment containing the antigen of interest or make use of a radioactive label. Immunofluorescent techniques that utilized labelled antibodies were conceptualized in the 1940s by Albert H. Coons.thumb|280x280px|right|Photomicrograph of a [[histology|histological section of human skin prepared for direct immunofluorescence using an anti-IgG antibody. The skin is from a patient with systemic lupus erythematosus and shows IgG deposit at two different places: The first is a band-like deposit along the epidermal basement membrane ("lupus band test" is positive). The second is within the nuclei of the epidermal cells (anti-nuclear antibodies).]]

Immunofluorescence is employed in foundational scientific investigations and clinical diagnostic endeavors, showcasing its multifaceted utility across diverse substrates, including tissue sections, cultured cell lines, or individual cells. Its usage includes analysis of the distribution of proteins, glycans, small biological and non-biological molecules, and visualization of structures such as intermediate-sized filaments.

If the topology of a cell membrane is undetermined, epitope insertion into proteins can be used in conjunction with immunofluorescence to determine structures within the cell membrane. Immunofluorescence (IF) can also be used as a "semi-quantitative" method to gain insight into the levels and localization patterns of DNA methylation. IF can additionally be used in combination with other, non-antibody methods of fluorescent staining, e.g., the use of DAPI to label DNA.

Examination of immunofluorescence specimens can be conducted utilizing various microscope configurations, including the epifluorescence microscope, confocal microscope, and widefield microscope.

Types

thumb|Main [[antinuclear antibody patterns on immunofluorescence.]]

Preparation of fluorescence

To perform immunofluorescence staining, a fluorophore must be conjugated ("tagged") to an antibody. Staining procedures can be applied to both retained intracellular expressed antibodies, or to cell surface antigens on living cells. There are two general classes of immunofluorescence techniques: primary (direct) and secondary (indirect).

The direct attachment of the fluorophore to the antibody reduces the number of steps in the sample preparation procedure, saving time and reducing non-specific background signal during analysis.

Limitations

thumb|257x257px|Basic concept of the ABC-method: Primary antibody binds to the antigen, before binding to the biotinylated secondary antibody. Avidin-Biotin enzyme complex (ABC) then attaches to the secondary antibody.

Immunofluorescence is only limited to fixed (i.e. dead) cells, when studying structures within the cell, as antibodies generally do not penetrate intact cellular or subcellular membranes in living cells, because they are large proteins. To visualize these structures, antigenic material must be fixed firmly on its natural localization inside the cell. To study structures within living cells, in combination with fluorescence, one can utilize recombinant proteins containing fluorescent protein domains, e.g., green fluorescent protein (GFP). The GFP-technique involves altering the genetic information of the cells.

A significant problem with immunofluorescence is photobleaching,

Advances

The main improvements to immunofluorescence lie in the development of fluorophores and fluorescent microscopes. Fluorophores can be structurally modified to improve brightness and photostability, while preserving spectral properties and cell permeability.

thumb|286x286px|Photomicrograph of a [[histology|histological section of human skin prepared for direct immunofluorescence using an anti-IgA antibody. The skin is from a patient with Henoch–Schönlein purpura: IgA deposits are found in the walls of small superficial capillaries (yellow arrows). The pale wavy green area on top is the epidermis, the bottom fibrous area is the dermis.]]

Super-resolution fluorescence microscopy methods can produce images with a higher resolution than those microscopes imposed by the diffraction limit. This enables the determination of structural details within the cell. Super-resolution in fluorescence, more specifically, refers to the ability of a microscope to prevent the simultaneous fluorescence of adjacent spectrally identical fluorophores (spectral overlap). Some of the recently developed super-resolution fluorescent microscope methods include stimulated emission depletion (STED) microscopy, saturated structured-illumination microscopy (SSIM), fluorescence photoactivation localization microscopy (FPALM), and stochastic optical reconstruction microscopy (STORM).

Notable people

<!---♦♦♦ Only add a person to this list if they already have their own article on the English Wikipedia ♦♦♦--->

<!---♦♦♦ Please keep the list in alphabetical order by LAST NAME ♦♦♦--->

  • Albert Hewett Coons (1912–1978), physician, pathologist and immunologist
  • Cornelia Mitchell Downs (1892–1987), microbiologist and journalist

See also

  • Antibodies
  • Cutaneous conditions with immunofluorescence findings
  • Fluorescence
  • Immunochemistry
  • Immunohistochemistry
  • Patching and Capping

References

  • Images associated with autoimmune diseases at University of Birmingham
  • Overview at Davidson College