Last updated S. cerevisiae septins revealed with fluorescent microscopy utilizing fluorescent labeling
In molecular biology and biotechnology, a fluorescent tag, also known as a fluorescent label or fluorescent probe, is a molecule that is attached chemically to aid in the detection of a biomolecule such as a protein, antibody, or amino acid. Generally, fluorescent tagging, or labeling, uses a reactive derivative of a fluorescent molecule known as a fluorophore. The fluorophore selectively binds to a specific region or functional group on the target molecule and can be attached chemically or biologically.[1] Various labeling techniques such as enzymatic labeling, protein labeling, and genetic labeling are widely utilized. Ethidium bromide, fluorescein and green fluorescent protein 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.[2]
Stokes George GOsamu Shimomura-press conference Dec 06th, 2008-1
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 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.[3] 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 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 in 1897 to describe a chemical group associated with fluorescence. Since then, Fluorescein 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 in 1911.[4]
Ethidium bromide and variants were developed in the 1950s,[4] and in 1994, fluorescent proteins or FPs were introduced.[5]Green fluorescent protein or GFP was discovered by Osamu Shimomura in the 1960s and was developed as a tracer molecule by Douglas Prasher in 1987.[6] FPs led to a breakthrough of live cell imaging with the ability to selectively tag genetic protein regions and observe protein functions and mechanisms.[5] For this breakthrough, Shimomura was awarded the Nobel Prize in 2008.[7]
New methods for tracking biomolecules have been developed including the use of colorimetric biosensors, photochromic compounds, biomaterials, 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.[1]
Types of biosensors
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.[8] These polypeptides are then put through mass spectrometry. 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.[9] 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.[10]
Colorimetric assays are normally used to determine how much concentration of one species there is relative to another.[9]
Photochromic compounds
Photochromic 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.[11]
The most common organic molecule to be used as a photochrome is diarylethene.[12] 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.[11]
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.[13]
Electrochemical sensors
Electrochemical sensors can be 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.[14] Fluorescent tags can be used in conjunction with electrochemical sensors for ease of detection in a biological system.
Fluorescent labels
Aequorea victoriaGFP structure
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.[1]
Green fluorescent protein 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 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, BFP or blue fluorescent protein, and CFP or cyan fluorescent protein are examples of GFP variants. These variants are produced by the genetic engineering of the GFP gene.[15]
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.[16] 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.[1]
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.[17]
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.[18]
For instance, FAST is a variant of photoactive yellow protein which was engineered to bind chemical mimics of the GFP tripeptide chromophore.[19] Likewise, the spinach aptamer is an engineered RNA sequence which can bind GFP chromophore chemical mimics, thereby conferring conditional and reversible fluorescence on RNA molecules containing the sequence.[20]
Use of tags in fluorescent labeling
In a direct fluorescent antibody test, antibodies have been chemically linked to a fluorescent dyeFISH image of bifidobacteria Cy3FISH analysis di george syndrome
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.[1] 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.[21] 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.[22] 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.[23] 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.[22]
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.[1]
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. 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.[24]
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.[25] Proteins can then be labeled and detected with imaging such as super-resolution microscopy, Ca2+-imaging, pH sensing, hydrogen peroxide detection, chromophore assisted light inactivation, and multi-photon light microscopy. In vivo imaging studies in live animals have been performed for the first time with the use of a monomeric protein derived from the bacterial haloalkane dehalogenase known as the Halo-tag.[22][26] The Halo-tag covalently links to its ligand and allows for better expression of soluble proteins.[26]
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.[27] Moreover, radiation and appropriate handling is no longer a concern.
With the development of fluorescent tagging, fluorescence microscopy 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.[24]
Latest advances in methods involving fluorescent tags have led to the visualization of mRNA 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 embryo localizes to the posterior region of the oocyte.[17]
↑ Chen X, Smith LM, Bradbury EM (March 2000). "Site-specific mass tagging with stable isotopes in proteins for accurate and efficient protein identification". Analytical Chemistry. 72 (6): 1134–43. doi:10.1021/ac9911600. PMID10740850.
↑ Halevy, Revital; Sofiya Kolusheval; Robert E.W. Hancock; Raz Jelinek (2002). "Colorimetric Biosensor Vesicles for Biotechnological Applications"(PDF). Materials Research Society Symposium Proceedings. 724. Biological and Biomimetic Materials - Properties to Function. Archived(PDF) from the original on October 14, 2013. Retrieved 4 April 2013.
↑ Perrier A, Maurel F, Jacquemin D (August 2012). "Single molecule multiphotochromism with diarylethenes". Accounts of Chemical Research. 45 (8): 1173–82. doi:10.1021/ar200214k. PMID22668009.
↑ Jung D, Min K, Jung J, Jang W, Kwon Y (May 2013). "Chemical biology-based approaches on fluorescent labeling of proteins in live cells". Molecular BioSystems. 9 (5): 862–72. doi:10.1039/c2mb25422k. PMID23318293.
↑ Szent-Gyorgyi C, Schmidt BF, Schmidt BA, Creeger Y, Fisher GW, Zakel KL, Adler S, Fitzpatrick JA, Woolford CA, Yan Q, Vasilev KV, Berget PB, Bruchez MP, Jarvik JW, Waggoner A (February 2008). "Fluorogen-activating single-chain antibodies for imaging cell surface proteins". Nature Biotechnology (Abstract). 26 (2): 235–40. doi:10.1038/nbt1368. PMID18157118. S2CID21815631. We report here the development of protein reporters that generate fluorescence from otherwise dark molecules (fluorogens).
↑ Jung D, Min K, Jung J, Jang W, Kwon Y (May 2013). "Chemical biology-based approaches on fluorescent labeling of proteins in live cells". Molecular BioSystems. 9 (5): 862–72. doi:10.1039/C2MB25422K. PMID23318293.
1 2 Matthew P Scott; Lodish, Harvey F.; Arnold Berk; Kaiser, Chris; Monty Krieger; Anthony Bretscher; Hidde Ploegh; Angelika Amon (2012). Molecular Cell Biology. San Francisco: W. H. Freeman. ISBN978-1-4292-3413-9.
The green fluorescent protein (GFP) is a protein that exhibits 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 Aequorea victoria and is sometimes called avGFP. However, GFPs have been found in other organisms including corals, sea anemones, zoanithids, copepods and lancelets.
A fluorophore 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 several π bonds.
Förster resonance energy transfer (FRET), fluorescence resonance energy transfer, resonance energy transfer (RET) or electronic energy transfer (EET) is a mechanism describing energy transfer between two light-sensitive molecules (chromophores). A donor chromophore, initially in its electronic excited state, may transfer energy to an acceptor chromophore through nonradiative dipole–dipole coupling. The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance.
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 microscope" refers to any microscope that uses fluorescence to generate an image, whether it is a simple set up like an epifluorescence microscope or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescence image.
Chemical biology is a scientific discipline between the fields of chemistry and biology. The discipline involves the application of chemical techniques, analysis, and often small molecules produced through synthetic chemistry, to the study and manipulation of biological systems. Although often confused with biochemistry, which studies the chemistry of biomolecules and regulation of biochemical pathways within and between cells, chemical biology remains distinct by focusing on the application of chemical tools to address biological questions.
Bimolecular fluorescence complementation is a technology typically used to validate protein interactions. It is based on the association of fluorescent protein fragments that are attached to components of the same macromolecular complex. Proteins that are postulated to interact are fused to unfolded complementary fragments of a fluorescent reporter protein and expressed in live cells. Interaction of these proteins will bring the fluorescent fragments within proximity, allowing the reporter protein to reform in its native three-dimensional structure and emit its fluorescent signal. This fluorescent signal can be detected and located within the cell using an inverted fluorescence microscope that allows imaging of fluorescence in cells. In addition, the intensity of the fluorescence emitted is proportional to the strength of the interaction, with stronger levels of fluorescence indicating close or direct interactions and lower fluorescence levels suggesting interaction within a complex. Therefore, through the visualisation and analysis of the intensity and distribution of fluorescence in these cells, one can identify both the location and interaction partners of proteins of interest.
Molecular imaging is a field of medical imaging that focuses on imaging molecules of medical interest within living patients. This is in contrast to conventional methods for obtaining molecular information from preserved tissue samples, such as histology. Molecules of interest may be either ones produced naturally by the body, or synthetic molecules produced in a laboratory and injected into a patient by a doctor. The most common example of molecular imaging used clinically today is to inject a contrast agent into a patient's bloodstream and to use an imaging modality to track its movement in the body. Molecular imaging originated from the field of radiology from a need to better understand fundamental molecular processes inside organisms in a noninvasive manner.
A molecular sensor or chemosensor is a molecular structure that is used for sensing of an analyte to produce a detectable change or a signal. The action of a chemosensor, relies on an interaction occurring at the molecular level, usually involves the continuous monitoring of the activity of a chemical species in a given matrix such as solution, air, blood, tissue, waste effluents, drinking water, etc. The application of chemosensors is referred to as chemosensing, which is a form of molecular recognition. All chemosensors are designed to contain a signalling moiety and a recognition moiety, that is connected either directly to each other or through a some kind of connector or a spacer. The signalling is often optically based electromagnetic radiation, giving rise to changes in either the ultraviolet and visible absorption or the emission properties of the sensors. Chemosensors may also be electrochemically based. Small molecule sensors are related to chemosensors. These are traditionally, however, considered as being structurally simple molecules and reflect the need to form chelating molecules for complexing ions in analytical chemistry. Chemosensors are synthetic analogues of biosensors, the difference being that biosensors incorporate biological receptors such as antibodies, aptamers or large biopolymers.
Fluorescence Loss in Photobleaching (FLIP) is a fluorescence microscopy technique used to examine movement of molecules inside cells and membranes. A cell membrane is typically labeled with a fluorescent dye to allow for observation. A specific area of this labeled section is then bleached several times using the beam of a confocal laser scanning microscope. After each imaging scan, bleaching occurs again. This occurs several times, to ensure that all accessible fluorophores are bleached since unbleached fluorophores are exchanged for bleached fluorophores, causing movement through the cell membrane. The amount of fluorescence from that region is then measured over a period of time to determine the results of the photobleaching on the cell as a whole.
EosFP is a photoactivatable green to red fluorescent protein. Its green fluorescence (516 nm) switches to red (581 nm) upon UV irradiation of ~390 nm due to a photo-induced modification resulting from a break in the peptide backbone near the chromophore. Eos was first discovered as a tetrameric protein in the stony coral Lobophyllia hemprichii. Like other fluorescent proteins, Eos allows for applications such as the tracking of fusion proteins, multicolour labelling and tracking of cell movement. Several variants of Eos have been engineered for use in specific study systems including mEos2, mEos4 and CaMPARI.
Fluorescence is used in the life sciences generally as a non-destructive way of tracking or analysing biological molecules. Some proteins or small molecules in cells are naturally fluorescent, which is called intrinsic fluorescence or autofluorescence. Alternatively, specific or general proteins, nucleic acids, lipids or small molecules can be "labelled" with an extrinsic fluorophore, a fluorescent dye which can be a small molecule, protein or quantum dot. Several techniques exist to exploit additional properties of fluorophores, such as fluorescence resonance energy transfer, where the energy is passed non-radiatively to a particular neighbouring dye, allowing proximity or protein activation to be detected; another is the change in properties, such as intensity, of certain dyes depending on their environment allowing their use in structural studies.
Microscale thermophoresis (MST) is a technology for the biophysical analysis of interactions between biomolecules. Microscale thermophoresis is based on the detection of a temperature-induced change in fluorescence of a target as a function of the concentration of a non-fluorescent ligand. The observed change in fluorescence is based on two distinct effects. On the one hand it is based on a temperature related intensity change (TRIC) of the fluorescent probe, which can be affected by binding events. On the other hand, it is based on thermophoresis, the directed movement of particles in a microscopic temperature gradient. Any change of the chemical microenvironment of the fluorescent probe, as well as changes in the hydration shell of biomolecules result in a relative change of the fluorescence detected when a temperature gradient is applied and can be used to determine binding affinities. MST allows measurement of interactions directly in solution without the need of immobilization to a surface.
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 techniques rely on the near-field or on the far-field. Among techniques that rely on the latter are those that improve the resolution only modestly beyond the diffraction-limit, such as confocal microscopy with closed pinhole or aided by computational methods such as deconvolution or detector-based pixel reassignment, the 4Pi microscope, and structured-illumination microscopy technologies such as SIM and SMI.
There are many methods to investigate protein–protein interactions which are the physical contacts of high specificity established between two or more protein molecules involving electrostatic forces and hydrophobic effects. Each of the approaches has its own strengths and weaknesses, especially with regard to the sensitivity and specificity of the method. A high sensitivity means that many of the interactions that occur are detected by the screen. A high specificity indicates that most of the interactions detected by the screen are occurring in reality.
Photo-activated localization microscopy and stochastic optical reconstruction microscopy (STORM) are widefield fluorescence microscopy imaging methods that allow obtaining images with a resolution beyond the diffraction limit. The methods were proposed in 2006 in the wake of a general emergence of optical super-resolution microscopy methods, and were featured as Methods of the Year for 2008 by the Nature Methods journal. The development of PALM as a targeted biophysical imaging method was largely prompted by the discovery of new species and the engineering of mutants of fluorescent proteins displaying a controllable photochromism, such as photo-activatible GFP. However, the concomitant development of STORM, sharing the same fundamental principle, originally made use of paired cyanine dyes. One molecule of the pair, when excited near its absorption maximum, serves to reactivate the other molecule to the fluorescent state.
In the field of cellular biology, single-cell analysis and subcellular analysis is the study of genomics, transcriptomics, proteomics, metabolomics and cell–cell interactions at the single cell level. The concept of single-cell analysis originated in the 1970s. Before the discovery of heterogeneity, single-cell analysis mainly referred to the analysis or manipulation of an individual cell in a bulk population of cells at a particular condition using optical or electronic microscope. To date, due to the heterogeneity seen in both eukaryotic and prokaryotic cell populations, analyzing a single cell makes it possible to discover mechanisms not seen when studying a bulk population of cells. Technologies such as fluorescence-activated cell sorting (FACS) allow the precise isolation of selected single cells from complex samples, while high throughput single cell partitioning technologies, enable the simultaneous molecular analysis of hundreds or thousands of single unsorted cells; this is particularly useful for the analysis of transcriptome variation in genotypically identical cells, allowing the definition of otherwise undetectable cell subtypes. The development of new technologies is increasing our ability to analyze the genome and transcriptome of single cells, as well as to quantify their proteome and metabolome. Mass spectrometry techniques have become important analytical tools for proteomic and metabolomic analysis of single cells. Recent advances have enabled quantifying thousands of protein across hundreds of single cells, and thus make possible new types of analysis. In situ sequencing and fluorescence in situ hybridization (FISH) do not require that cells be isolated and are increasingly being used for analysis of tissues.
FlAsH-EDT2 is an organoarsenic compound with molecular formula C24H18As2O5S4. Its structure is based around a fluorescein core with two 1,3,2-dithiarsolane substituents. It is used in bioanalytical research as a fluorescent label for visualising proteins in living cells. FlAsH-EDT2 is an abbreviation for fluorescin arsenical hairpin binder-ethanedithiol, and is a pale yellow or pinkish fluorogenic solid. It has a semi-structural formula (C2H4AsS2)2-(C13H5O3)-C6H4COOH, representing the dithiarsolane substituents bound to the hydroxyxanthone core, attached to an o-substituted molecule of benzoic acid.
PRIME is a molecular biology research tool developed by Alice Y. Ting and the Ting Lab at MIT for site-specific labeling of proteins in living cells with chemical probes. Probes often have useful biophysical properties, such as fluorescence, and allow imaging of proteins. Ultimately, PRIME enables scientists to study functions of specific proteins of interest.
Fluorescence imaging is a type of non-invasive imaging technique that can help visualize biological processes taking place in a living organism. Images can be produced from a variety of methods including: microscopy, imaging probes, and spectroscopy.
FAST is a small, genetically-encoded, protein tag which allows for fluorescence reporting of proteins of interest. Unlike natural fluorescent proteins and derivates such as GFP or mCherry, FAST is not fluorescent by itself. It can bind selectively a fluorogenic chromophore derived from 4-hydroxybenzylidene rhodanine (HBR), which is itself non fluorescent unless bound. Once bound, the pair of molecules goes through a unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, hence providing high labeling selectivity. The FAST-fluorogen reporting system can be used in fluorescence microscopy, flow cytometry and any other fluorometric method to explore the living world: biosensors, protein trafficking.
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