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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, than the absorbed radiation. A perceptible example of fluorescence occurs when the absorbed radiation is in the ultraviolet region of the electromagnetic spectrum, while the emitted light is in the visible region; this gives the fluorescent substance a distinct color that can only be seen when the substance has been exposed to UV light. Fluorescent materials cease to glow nearly immediately when the radiation source stops, unlike phosphorescent materials, which continue to emit light for some time after.
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.
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.
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.
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 is a useful tool in cell and molecular biology because the excitation and emission peaks of YFP are distinguishable from GFP which allows for the study of multiple processes/proteins within the same experiment.
Roger Yonchien Tsien was an American biochemist. He was a professor of chemistry and biochemistry at the University of California, San Diego and was awarded the Nobel Prize in Chemistry in 2008 for his discovery and development of the green fluorescent protein, in collaboration with organic chemist Osamu Shimomura and neurobiologist Martin Chalfie. Tsien was also a pioneer of calcium imaging.
Kaede is a photoactivatable fluorescent protein naturally originated from a stony coral, Trachyphyllia geoffroyi. Its name means "maple" in Japanese. With the irradiation of ultraviolet light (350–400 nm), Kaede undergoes irreversible photoconversion from green fluorescence to red fluorescence.
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.
Photoactivatable fluorescent proteins (PAFPs) is a type of fluorescent protein that exhibit fluorescence that can be modified by a light-induced chemical reaction.
Vertico spatially modulated illumination (Vertico-SMI) is the fastest light microscope for the 3D analysis of complete cells in the nanometer range. It is based on two technologies developed in 1996, SMI and SPDM. The effective optical resolution of this optical nanoscope has reached the vicinity of 5 nm in 2D and 40 nm in 3D, greatly surpassing the λ/2 resolution limit applying to standard microscopy using transmission or reflection of natural light according to the Abbe resolution limit That limit had been determined by Ernst Abbe in 1873 and governs the achievable resolution limit of microscopes using conventional techniques.
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.
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.
GCaMP is a genetically encoded calcium indicator (GECI) initially developed in 2001 by Junichi Nakai. It is a synthetic fusion of green fluorescent protein (GFP), calmodulin (CaM), and M13, a peptide sequence from myosin light-chain kinase. When bound to Ca2+, GCaMP fluoresces green with a peak excitation wavelength of 480 nm and a peak emission wavelength of 510 nm. It is used in biological research to measure intracellular Ca2+ levels both in vitro and in vivo using virally transfected or transgenic cell and animal lines. The genetic sequence encoding GCaMP can be inserted under the control of promoters exclusive to certain cell types, allowing for cell-type specific expression of GCaMP. Since Ca2+ is a second messenger that contributes to many cellular mechanisms and signaling pathways, GCaMP allows researchers to quantify the activity of Ca2+-based mechanisms and study the role of Ca2+ ions in biological processes of interest.
mCherry is a member of the mFruits family of monomeric red fluorescent proteins (mRFPs). As a RFP, mCherry was derived from DsRed of Discosoma sea anemones unlike green fluorescent proteins (GFPs) which are often derived from Aequorea victoria jellyfish. Fluorescent proteins are used to tag components in the cell, so they can be studied using fluorescence spectroscopy and fluorescence microscopy. mCherry absorbs light between 540-590 nm and emits light in the range of 550-650 nm. mCherry belongs to the group of fluorescent protein chromophores used as instruments to visualize genes and analyze their functions in experiments. Genome editing has been improved greatly through the precise insertion of these fluorescent protein tags into the genetic material of many diverse organisms. Most comparisons between the brightness and photostability of different fluorescent proteins have been made in vitro, removed from biological variables that affect protein performance in cells or organisms. It is hard to perfectly simulate cellular environments in vitro, and the difference in environment could have an effect on the brightness and photostability.
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 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.
Small ultra red fluorescent protein (smURFP) is a class of far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein, α-allophycocyanin. Native α-allophycocyanin requires an exogenous protein, known as a lyase, to attach the chromophore, phycocyanobilin. Phycocyanobilin is not present in mammalian cells. smURFP was evolved to covalently attach phycocyanobilin without a lyase and fluoresce, covalently attach biliverdin and fluoresce, blue-shift fluorescence to match the organic fluorophore, Cy5, and not inhibit E. coli growth. smURFP was found after 12 rounds of random mutagenesis and manually screening 10,000,000 bacterial colonies.
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.
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.
References
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- ↑ Miyawaki, Atsushi; Shcherbakova, Daria M; Verkhusha, Vladislav V (October 2012). "Red fluorescent proteins: chromophore formation and cellular applications". Current Opinion in Structural Biology. 22 (5): 679–688. doi:10.1016/j.sbi.2012.09.002. ISSN 0959-440X. PMC 3737244 . PMID 23000031.
- 1 2 Remington, S. James (1 January 2002). "Negotiating the speed bumps to fluorescence". Nature Biotechnology. 20 (1): 28–29. doi:10.1038/nbt0102-28. PMID 11753356. S2CID 37021603.
- 1 2 3 Piatkevich, Kiryl D.; Verkhusha, Vladislav V. (2011). "Guide to Red Fluorescent Proteins and Biosensors for Flow Cytometry". Methods in Cell Biology. 102: 431–461. doi:10.1016/B978-0-12-374912-3.00017-1. ISBN 9780123749123. ISSN 0091-679X. PMC 3987785 . PMID 21704849.
- ↑ Bindels, Daphne S; Haarbosch, Lindsay (2017). "mScarlet: a bright monomeric red fluorescent protein for cellular imaging". Nature Methods. 14 (1): 53–56. doi:10.1038/nmeth.4074. ISSN 1548-7105. PMID 27869816. S2CID 3539874.
- ↑ Böhm I, Gehrke S, Kleb B, Hungerbühler M, Müller R, Klose KJ, Alfke H (2019). "Monitoring of tumor burden in vivo by optical imaging in a xenograft SCID mouse model: evaluation of two fluorescent proteins of the GFP-superfamily". Acta Radiol. 60 (3): 315–326. doi:10.1177/0284185118780896. PMID 29890843. S2CID 48353442.