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molecular biology Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and phys ...
and
biotechnology Biotechnology is the integration of natural sciences and engineering sciences in order to achieve the application of organisms, cells, parts thereof and molecular analogues for products and services. The term ''biotechnology'' was first used ...
, a fluorescent tag, also known as a fluorescent label or fluorescent probe, is a
molecule A molecule is a group of two or more atoms held together by attractive forces known as chemical bonds; depending on context, the term may or may not include ions which satisfy this criterion. In quantum physics, organic chemistry, and b ...
that is attached chemically to aid in the detection of a
biomolecule A biomolecule or biological molecule is a loosely used term for molecules present in organisms that are essential to one or more typically biological processes, such as cell division, morphogenesis, or developmental biology, development. Biom ...
such as a protein, antibody, or amino acid. Generally,
fluorescent Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, ...
tagging, or labeling, uses a reactive derivative of a fluorescent molecule known as a
fluorophore A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with se ...
. The fluorophore selectively binds to a specific region or functional group on the target molecule and can be attached chemically or biologically. Various labeling techniques such as enzymatic labeling, protein labeling, and genetic labeling are widely utilized.
Ethidium bromide Ethidium bromide (or homidium bromide, chloride salt homidium chloride) is an intercalating agent commonly used as a fluorescent tag ( nucleic acid stain) in molecular biology laboratories for techniques such as agarose gel electrophoresis. It ...
,
fluorescein Fluorescein is an organic compound and dye based on the xanthene tricyclic structural motif, formally belonging to triarylmethine dyes family. It is available as a dark orange/red powder slightly soluble in water and alcohol. It is widely used ...
and
green fluorescent protein The green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. The label ''GFP'' traditionally refers to the protein first isolated from the jellyfish '' Aequore ...
are common tags. The most commonly labelled molecules are antibodies, proteins, amino acids and peptides which are then used as specific probes for detection of a particular target.


History

The development of methods to detect and identify biomolecules has been motivated by the ability to improve the study of molecular structure and interactions. Before the advent of fluorescent labeling,
radioisotopes A radionuclide (radioactive nuclide, radioisotope or radioactive isotope) is a nuclide that has excess nuclear energy, making it unstable. This excess energy can be used in one of three ways: emitted from the nucleus as gamma radiation; transferr ...
were used to detect and identify molecular compounds. Since then, safer methods have been developed that involve the use of fluorescent dyes or fluorescent proteins as tags or probes as a means to label and identify biomolecules. Although fluorescent tagging in this regard has only been recently utilized, the discovery of fluorescence has been around for a much longer time.
Sir George Stokes Sir George Gabriel Stokes, 1st Baronet, (; 13 August 1819 – 1 February 1903) was an Irish English physicist and mathematician. Born in County Sligo, Ireland, Stokes spent all of his career at the University of Cambridge, where he was the Luc ...
developed the Stokes Law of Fluorescence in 1852 which states that the wavelength of fluorescence emission is greater than that of the exciting radiation. Richard Meyer then termed
fluorophore A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with se ...
in 1897 to describe a chemical group associated with fluorescence. Since then,
Fluorescein Fluorescein is an organic compound and dye based on the xanthene tricyclic structural motif, formally belonging to triarylmethine dyes family. It is available as a dark orange/red powder slightly soluble in water and alcohol. It is widely used ...
was created as a fluorescent dye by Adolph von Baeyer in 1871 and the method of staining was developed and utilized with the development of
fluorescence microscopy A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microscop ...
in 1911. Ethidium bromide and variants were developed in the 1950s, and in 1994, fluorescent proteins or FPs were introduced.
Green fluorescent protein The green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. The label ''GFP'' traditionally refers to the protein first isolated from the jellyfish '' Aequore ...
or GFP was discovered by
Osamu Shimomura was a Japanese organic chemist and marine biologist, and Professor Emeritus at Marine Biological Laboratory (MBL) in Woods Hole, Massachusetts and Boston University School of Medicine. He was awarded the Nobel Prize in Chemistry in 2008 for th ...
in the 1960s and was developed as a tracer molecule by Douglas Prasher in 1987. FPs led to a breakthrough of live
cell imaging 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 ...
with the ability to selectively tag genetic protein regions and observe protein functions and mechanisms. For this breakthrough, Shimomura was awarded the Nobel Prize in 2008. New methods for tracking biomolecules have been developed including the use of colorimetric biosensors, photochromic compounds,
biomaterials A biomaterial is a substance that has been engineered to interact with biological systems for a medical purpose, either a therapeutic (treat, augment, repair, or replace a tissue function of the body) or a diagnostic one. As a science, biomateria ...
, and electrochemical sensors. Fluorescent labeling is also a common method in which applications have expanded to enzymatic labeling, chemical labeling, protein labeling, and genetic labeling.


Methods for tracking biomolecules

There are currently several labeling methods for tracking biomolecules. Some of the methods include the following.


Isotope markers

Common species that isotope markers are used for include proteins. In this case, amino acids with stable isotopes of either carbon, nitrogen, or hydrogen are incorporated into polypeptide sequences. These polypeptides are then put through
mass spectrometry Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a '' mass spectrum'', a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is u ...
. Because of the exact defined change that these isotopes incur on the peptides, it is possible to tell through the spectrometry graph which peptides contained the isotopes. By doing so, one can extract the protein of interest from several others in a group. Isotopic compounds play an important role as photochromes, described below.


Colorimetric biosensors

Biosensors are attached to a substance of interest. Normally, this substance would not be able to absorb light, but with the attached biosensor, light can be absorbed and emitted on a spectrophotometer. Additionally, biosensors that are fluorescent can be viewed with the naked eye. Some fluorescent biosensors also have the ability to change color in changing environments (ex: from blue to red). A researcher would be able to inspect and get data about the surrounding environment based on what color he or she could see visibly from the biosensor-molecule hybrid species. Colorimetric assays are normally used to determine how much concentration of one species there is relative to another.


Photochromic compounds

Photochromic Photochromism is the reversible transformation of a chemical species ( photoswitch) between two forms by the absorption of electromagnetic radiation ( photoisomerization), where the two forms have different absorption spectra. In plain language, ...
compounds have the ability to switch between a range or variety of colors. Their ability to display different colors lies in how they absorb light. Different isomeric manifestations of the molecule absorbs different wavelengths of light, so that each isomeric species can display a different color based on its absorption. These include photoswitchable compounds, which are proteins that can switch from a non-fluorescent state to that of a fluorescent one given a certain environment. The most common organic molecule to be used as a photochrome is
diarylethene Diarylethene is the general name of a class of chemical compounds that have aromatic functional groups bonded to each end of a carbon–carbon double bond. The simplest example is stilbene, which has two geometric isomers, E and Z. Under the influ ...
. Other examples of photoswitchable proteins include PADRON-C, rs-FastLIME-s and bs-DRONPA-s, which can be used in plant and mammalian cells alike to watch cells move into different environments.


Biomaterials

Fluorescent biomaterials are a possible way of using external factors to observe a pathway more visibly. The method involves fluorescently labeling peptide molecules that would alter an organism's natural pathway. When this peptide is inserted into the organism's cell, it can induce a different reaction. This method can be used, for example to treat a patient and then visibly see the treatment's outcome.


Electrochemical sensors

Electrochemical sensors can used for label-free sensing of biomolecules. They detect changes and measure current between a probed metal electrode and an electrolyte containing the target analyte. A known potential to the electrode is then applied from a feedback current and the resulting current can be measured. For example, one technique using electrochemical sensing includes slowly raising the voltage causing chemical species at the electrode to be oxidized or reduced. Cell current vs voltage is plotted which can ultimately identify the quantity of chemical species consumed or produced at the electrode. Fluorescent tags can be used in conjunction with electrochemical sensors for ease of detection in a biological system.


Fluorescent labels

Of the various methods of labeling biomolecules, fluorescent labels are advantageous in that they are highly sensitive even at low concentration and non-destructive to the target molecule folding and function.
Green fluorescent protein The green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. The label ''GFP'' traditionally refers to the protein first isolated from the jellyfish '' Aequore ...
is a naturally occurring fluorescent protein from the jellyfish ''Aequorea victoria'' that is widely used to tag proteins of interest. GFP emits a photon in the green region of the light spectrum when excited by the absorption of light. The
chromophore A chromophore is the part of a molecule responsible for its color. The color that is seen by our eyes is the one not absorbed by the reflecting object within a certain wavelength spectrum of visible light. The chromophore is a region in the mo ...
consists of an oxidized tripeptide -Ser^65-Tyr^66-Gly^67 located within a β barrel. GFP catalyzes the oxidation and only requires molecular oxygen. GFP has been modified by changing the wavelength of light absorbed to include other colors of fluorescence. YFP or
yellow fluorescent protein Yellow fluorescent protein (YFP) is a genetic mutant of green fluorescent protein (GFP) originally derived from the jellyfish '' Aequorea victoria''. Its excitation peak is 513 nm and its emission peak is 527 nm. Like the parent GFP, YFP ...
, BFP or
blue fluorescent protein The green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. The label ''GFP'' traditionally refers to the protein first isolated from the jellyfish ''Aequore ...
, and CFP or
cyan fluorescent protein The green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. The label ''GFP'' traditionally refers to the protein first isolated from the jellyfish '' Aequore ...
are examples of GFP variants. These variants are produced by the genetic engineering of the GFP gene. Synthetic fluorescent probes can also be used as fluorescent labels. Advantages of these labels include a smaller size with more variety in color. They can be used to tag proteins of interest more selectively by various methods including chemical recognition-based labeling, such as utilizing metal-chelating peptide tags, and biological recognition-based labeling utilizing enzymatic reactions. However, despite their wide array of excitation and emission wavelengths as well as better stability, synthetic probes tend to be toxic to the cell and so are not generally used in cell imaging studies. Fluorescent labels can be hybridized to mRNA to help visualize interaction and activity, such as mRNA localization. An antisense strand labeled with the fluorescent probe is attached to a single mRNA strand, and can then be viewed during cell development to see the movement of mRNA within the cell.


Fluorogenic labels

A fluorogen is a ligand (fluorogenic ligand) which is not itself fluorescent, but when it is bound by a specific protein or RNA structure becomes fluorescent. For instance,
FAST Fast or FAST may refer to: * Fast (noun), high speed or velocity * Fast (noun, verb), to practice fasting, abstaining from food and/or water for a certain period of time Acronyms and coded Computing and software * ''Faceted Application of Subje ...
is a variant of photoactive yellow protein which was engineered to bind chemical mimics of the GFP tripeptide chromophore. Likewise, the
spinach aptamer The need for fluorescently tracking RNA rose as its roles in complex cellular functions has grown to not only include mRNA, rRNA, and tRNA, but also RNAi, siRNA, snoRNA, and lncRNA, among others. Spinach is a synthetically derived RNA aptamer bo ...
is an engineered RNA sequence which can bind GFP chromophore chemical mimics, thereby conferring conditional and reversible fluorescence on RNA molecules containing the sequence.


Use of tags in fluorescent labeling

Fluorescent labeling is known for its non-destructive nature and high sensitivity. This has made it one of the most widely used methods for labeling and tracking biomolecules. Several techniques of fluorescent labeling can be utilized depending on the nature of the target.


Enzymatic labeling

In enzymatic labeling, a DNA construct is first formed, using a gene and the DNA of a fluorescent protein. After transcription, a hybrid RNA + fluorescent is formed. The object of interest is attached to an enzyme that can recognize this hybrid DNA. Usually fluorescein is used as the fluorophore.


Chemical labeling

Chemical labeling or the use of chemical tags utilizes the interaction between a small molecule and a specific genetic amino acid sequence. Chemical labeling is sometimes used as an alternative for GFP. Synthetic proteins that function as fluorescent probes are smaller than GFP's, and therefore can function as probes in a wider variety of situations. Moreover, they offer a wider range of colors and photochemical properties. With recent advancements in chemical labeling, Chemical tags are preferred over fluorescent proteins due to the architectural and size limitations of the fluorescent protein's characteristic β-barrel. Alterations of fluorescent proteins would lead to loss of fluorescent properties.


Protein labeling

Protein labeling use a short tag to minimize disruption of protein folding and function. Transition metals are used to link specific residues in the tags to site-specific targets such as the N-termini, C-termini, or internal sites within the protein. Examples of tags used for protein labeling include biarsenical tags, Histidine tags, and FLAG tags.


Genetic labeling

Fluorescence in situ hybridization (FISH), is an example of a genetic labeling technique that utilizes probes that are specific for chromosomal sites along the length of a chromosome, also known as
chromosome painting Fluorescence ''in situ'' hybridization (FISH) is a molecular cytogenetic technique that uses fluorescent probes that bind to only particular parts of a nucleic acid sequence with a high degree of sequence complementarity. It was developed ...
. Multiple fluorescent dyes that each have a distinct excitation and emission wavelength are bound to a probe which is then hybridized to chromosomes. A fluorescence microscope can detect the dyes present and send it to a computer that can reveal the karyotype of a cell. This technique allows abnormalities such as deletions and duplications to be revealed.


Cell imaging

Chemical tags have been tailored for imaging technologies more so than fluorescent proteins because chemical tags can localize photosensitizers closer to the target proteins. Proteins can then be labeled and detected with imaging such as
super-resolution microscopy Super-resolution microscopy is a series of techniques in optical microscopy that allow such images to have resolutions higher than those imposed by the diffraction limit, which is due to the diffraction of light. Super-resolution imaging techni ...
, Ca2+-imaging, pH sensing, hydrogen peroxide detection, chromophore assisted light inactivation, and multi-photon light microscopy.
In vivo Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and p ...
imaging studies in live animals have been performed for the first time with the use of a
monomeric In chemistry, a monomer ( ; ''mono-'', "one" + '' -mer'', "part") is a molecule that can react together with other monomer molecules to form a larger polymer chain or three-dimensional network in a process called polymerization. Classification Mo ...
protein derived from the bacterial haloalkane dehalogenase known as the Halo-tag. The Halo-tag
covalently A covalent bond is a chemical bond that involves the sharing of electrons to form electron pairs between atoms. These electron pairs are known as shared pairs or bonding pairs. The stable balance of attractive and repulsive forces between atom ...
links to its
ligand In coordination chemistry, a ligand is an ion or molecule (functional group) that binds to a central metal atom to form a coordination complex. The bonding with the metal generally involves formal donation of one or more of the ligand's elect ...
and allows for better expression of soluble proteins.


Advantages

Although fluorescent dyes may not have the same sensitivity as radioactive probes, they are able to show real-time activity of molecules in action. Moreover, radiation and appropriate handling is no longer a concern. With the development of fluorescent tagging,
fluorescence microscopy A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microscop ...
has allowed the visualization of specific proteins in both fixed and live cell images. Localization of specific proteins has led to important concepts in cellular biology such as the functions of distinct groups of proteins in cellular membranes and organelles. In live cell imaging, fluorescent tags enable movements of proteins and their interactions to be monitored. Latest advances in methods involving fluorescent tags have led to the visualization of
mRNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein. mRNA is created during the ...
and its localization within various organisms. Live cell imaging of RNA can be achieved by introducing synthesized RNA that is chemically coupled with a fluorescent tag into living cells by microinjection. This technique was used to show how the ''oskar'' mRNA in the ''
Drosophila ''Drosophila'' () is a genus of flies, belonging to the family Drosophilidae, whose members are often called "small fruit flies" or (less frequently) pomace flies, vinegar flies, or wine flies, a reference to the characteristic of many speci ...
'' embryo localizes to the posterior region of the
oocyte An oocyte (, ), oöcyte, or ovocyte is a female gametocyte or germ cell involved in reproduction. In other words, it is an immature ovum, or egg cell. An oocyte is produced in a female fetus in the ovary during female gametogenesis. The female ...
.


See also

* Molecular tagging velocimetry *
Spectrophotometer for Nucleic Acid Measurements In molecular biology, quantitation of nucleic acids is commonly performed to determine the average concentrations of DNA or RNA present in a mixture, as well as their purity. Reactions that use nucleic acids often require particular amounts and p ...
*
Protein tag Protein tags are peptide sequences genetically grafted onto a recombinant protein. Tags are attached to proteins for various purposes. They can be added to either end of the target protein, so they are either C-terminus or N-terminus specific or a ...
s


Notes


External links

{{DEFAULTSORT:Fluorescent Tag Molecular biology Fluorescence techniques