Immunofluorescence
Immunofluorescence (IF) is a
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, do not interfere with the immunological specificity of the antibody or the binding capacity of its antigen.[4][5]
Immunofluorescence is a widely used example of
Immunofluorescence is employed in foundational scientific investigations and clinical diagnostic endeavors, showcasing its multifaceted utility across diverse substrates, including tissue sections, cultured
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.[9] 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.[10][11]
Examination of immunofluorescence specimens can be conducted utilizing various microscope configurations, including the
Types
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).[1][2] The following descriptions will focus primarily on these classes in terms of conjugated antibodies.[12]
Primary (direct)
Primary (direct) immunofluorescence (DIF) uses a single antibody, conjugated to a fluorophore. The antibody recognizes the target molecule (antigen) and binds to a specific region, called the epitope. The attached fluorophore can be detected via fluorescent microscopy, which, depending on the type of fluorophore, will emit a specific wavelength of light once excited.[1][14]
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.[12] This also limits the possibility of antibody cross-reactivity, and possible mistakes throughout the process. One disadvantage of DIF is the limited number of antibodies that can bind to the antigen. This limitation may reduce sensitivity to the technique. When the target protein is available in only small concentrations, a better approach would be secondary IF, which is considered to be more sensitive than DIF [2][12] when compared to Secondary (Indirect) Immunofluorescence.[1]
Secondary (indirect)
Secondary (indirect) immunofluorescence (SIF) is similar to direct immunofluorescence, however the technique utilizes two types of antibodies whereas only one of them have a conjugated fluorophore. The antibody with the conjugated fluorophore is referred to as the secondary antibody, while the unconjugated is referred to as the primary antibody.[1]
The principle of this technique is that the primary antibody specifically binds to the epitope on the target molecule, whereas the secondary antibody, with the conjugated fluorophore, recognizes and binds to the primary antibody.[1]
This technique is considered to be more sensitive than primary immunofluorescence, because multiple secondary antibodies can bind to the same primary antibody. The increased number of fluorophore molecules per antigen increases the amount of emitted light, and thus amplifies the signal.[1] There are different methods for attaining a higher fluorophore-antigen ratio such as the Avidin-Biotin Complex (ABC method) and Labeled Streptavidin-Biotin (LSAB method).[15][16]
Limitations
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.
A significant problem with immunofluorescence is photobleaching,[12] the fluorophores permanent loss of ability to emit light.[1] To mitigate the risk of photobleaching one can employ different strategies. By reducing or limiting the intensity, or timespan of light exposure, the absorption-emission cycle of fluorescent light is decreased, thus preserving the fluorophores functionality. One can also increase the concentration of fluorophores, or opt for more robust fluorophores that exhibit resilience against photobleaching such as Alexa Fluors, Seta Fluors, or DyLight Fluors.[2]
Other problems that may arise when using immunofluorescence techniques include autofluorescence, spectral overlap and non-specific staining.[1][2] Autofluorescence includes the natural fluorescence emitted from the sample tissue or cell itself. Spectral overlap happens when a fluorophore has a broad emission specter, that overlaps with the specter of another fluorophore, thus giving rise to false signals. Non-specific staining occurs when the antibody, containing the fluorophore, binds to unintended proteins because of sufficient similarity in the epitope. This can lead to false positives.[2][4][1]
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.[20]
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.[21] 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).[22]
Notable people
- immunologist.
- Cornelia Mitchell Downs (1892–1987), microbiologist and journalist
See also
- Antibodies
- Cutaneous conditions with immunofluorescence findings
- Fluorescence
- Immunochemistry
- Immunohistochemistry
- Patching and Capping
References
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- ^ ISBN 978-0-12-803077-6, retrieved 2024-02-14
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- .
- ^ PMID 35163349.
- PMID 35359932.
Minor edits by Mikael Häggström, MD
- Attribution 4.0 International (CC BY 4.0) license - ^ "Immunohistochemical Staining Methods" (PDF). IHC Guidebook (Sixth ed.). Dako Denmark A/S, An Agilent Technologies Company. 2013. Archived from the original (PDF) on 2016-08-03. Retrieved 2014-05-14.
- PMID 30539454
- PMID 25826597.
- ^ "Fixation and Permeabilization in in[sic] Immunocytochemistry/Immunofluorescence (ICC/IF)". Novus Biologicals. 2024-02-14. Retrieved 2024-02-14.
- PMID 14611963.
- S2CID 3944607.
- PMID 25599551.
- PMID 19489737.
- S2CID 5545465.
External links
- Images associated with autoimmune diseases Archived 2009-09-25 at the Wayback Machine at University of Birmingham
- Overview at Davidson College
- Immunofluorescence at the U.S. National Library of Medicine Medical Subject Headings (MeSH)