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.
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. 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.
The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (also known as AMPA receptor, AMPAR, or quisqualate receptor) is an ionotropic transmembrane receptor for glutamate (iGluR) and predominantly Na+ ion channel that mediates fast synaptic transmission in the central nervous system (CNS). It has been traditionally classified as a non-NMDA-type receptor, along with the kainate receptor. Its name is derived from its ability to be activated by the artificial glutamate analog AMPA. The receptor was first named the "quisqualate receptor" by Watkins and colleagues after a naturally occurring agonist quisqualate and was only later given the label "AMPA receptor" after the selective agonist developed by Tage Honore and colleagues at the Royal Danish School of Pharmacy in Copenhagen. The GRIA2-encoded AMPA receptor ligand binding core (GluA2 LBD) was the first glutamate receptor ion channel domain to be crystallized.
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.
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.
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.
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.
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.
Brainbow is a process by which individual neurons in the brain can be distinguished from neighboring neurons using fluorescent proteins. By randomly expressing different ratios of red, green, and blue derivatives of green fluorescent protein in individual neurons, it is possible to flag each neuron with a distinctive color. This process has been a major contribution to the field of neural connectomics.
Axon terminals are distal terminations of the branches of an axon. An axon, also called a nerve fiber, is a long, slender projection of a nerve cell that conducts electrical impulses called action potentials away from the neuron's cell body to transmit those impulses to other neurons, muscle cells, or glands. Most presynaptic terminals in the central nervous system are formed along the axons, not at their ends.
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.
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.
Fluorescent chloride sensors are used for chemical analysis. The discoveries of chloride (Cl−) participations in physiological processes stimulates the measurements of intracellular Cl− in live cells and the development of fluorescent tools referred below.
Calcium imaging is a microscopy technique to optically measure the calcium (Ca2+) status of an isolated cell, tissue or medium. Calcium imaging takes advantage of calcium indicators, fluorescent molecules that respond to the binding of Ca2+ ions by fluorescence properties. Two main classes of calcium indicators exist: chemical indicators and genetically encoded calcium indicators (GECI). This technique has allowed studies of calcium signalling in a wide variety of cell types. In neurons, action potential generation is always accompanied by rapid influx of Ca2+ ions. Thus, calcium imaging can be used to monitor the electrical activity in hundreds of neurons in cell culture or in living animals, which has made it possible to observe the activity of neuronal circuits during ongoing behavior.
A FMN-binding fluorescent protein (FbFP), also known as a LOV-based fluorescent protein, is a small, oxygen-independent fluorescent protein that binds flavin mononucleotide (FMN) as a chromophore.
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 born out of the need for a way of studying the role of RNAs at the cellular level. This aptamer was created using Systematic Evolution for Ligands by EXponential enrichment, or SELEX, which is also known as in vitro evolution.
Red fluorescent protein (RFP) is a protein which acts as a fluorophore, fluorescing red-orange when excited. The original variant occurs naturally in the coral genus Discosoma, and is named DsRed. Several new variants have been developed using directed mutagenesis which fluoresce orange, red, and far-red.
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 genetically-encoded protein tag which, upon reversible combination with a fluorogenic chromophore, allows the reporting of proteins of interest. FAST, a small 14 kDa protein, was engineered from the photoactive yellow protein (PYP) by directed evolution. It was disclosed for the first time in 2016 by researchers from Ecole normale supérieure de Paris. FAST was further evolved into splitFAST (2019), a complementation system for protein-protein interaction monitoring, and CATCHFIRE (2023), a self-reporting protein dimerizing system.
Optogenetics began with methods to alter neuronal activity with light, using e.g. channelrhodopsins. In a broader sense, optogenetic approaches also include the use of genetically encoded biosensors to monitor the activity of neurons or other cell types by measuring fluorescence or bioluminescence. Genetically encoded calcium indicators (GECIs) are used frequently to monitor neuronal activity, but other cellular parameters such as membrane voltage or second messenger activity can also be recorded optically. The use of optogenetic sensors is not restricted to neuroscience, but plays increasingly important roles in immunology, cardiology and cancer research.
