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The Alexa Fluor family of fluorescent dyes is a series of dyes invented by Molecular Probes, now a part of Thermo Fisher Scientific, and sold under the Invitrogen brand name. Alexa Fluor dyes are frequently used as cell and tissue labels in fluorescence microscopy and cell biology. [1] Alexa Fluor dyes can be conjugated directly to primary antibodies or to secondary antibodies to amplify signal and sensitivity [2] or other biomolecules.[ citation needed ]
The excitation and emission spectra of the Alexa Fluor series cover the visible spectrum and extend into the infrared. [3] The individual members of the family are numbered according roughly to their excitation maxima in nanometers.
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Richard and Rosaria Haugland, the founders of Molecular Probes, are well known in biology and chemistry for their research into fluorescent dyes useful in biological research applications.[ according to whom? ] At the time that Molecular Probes was founded,[ when? ] such products were largely unavailable commercially.[ citation needed ] A number of fluorescent dyes that are now widely used were discovered and developed in the laboratories of Molecular Probes.[ citation needed ]—dyes such as Texas Red, Cascade Blue, Oregon Green, Marina Blue, and the Alexa Fluor family.[ citation needed ] The most famous of these, the Alexa Fluor family of dyes,[ according to whom? ] were designed to improve upon properties of previously developed biological fluorescent dye families, and solve some of the issues that they possessed.[ citation needed ] The Alexa Fluor dyes were named after Alex Haugland, son of Richard and Rosaria Haugland.[ citation needed ]
Molecular Probes was acquired in 2003 by Invitrogen, [4] who worked to further expand the Alexa Fluor family by the addition of new dyes to fill gaps not covered in the emission spectrum.[ citation needed ] In 2008, Invitrogen and the Alexa Fluor product line became a part of the Life Technologies, after the Invitrogen merger with Applied Biosystems.[ citation needed ] In 2014, Life Technologies was acquired by Thermo Fisher Scientific, who revitalized the Invitrogen name and brand, bringing the Alexa Fluor product line back under it.[ citation needed ]
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The Alexa Fluor dyes were chemically synthesized through sulfonation and additional chemical modifications made to the well known families of coumarin, rhodamine, and cyanine dyes, and to the xanthene family (of which the fluorescein, an industry-standard, is a part).[ citation needed ] Sulfonation made the product Alexa Fluor dyes negatively charged and thus more hydrophilic and soluble than their parent dyes;[ citation needed ] the additional modifications were aimed to improve dye performance in other areas.[ citation needed ] For example, Alexa Fluor 488, a sulfonated and otherwise chemically modified[ clarification needed ] form of fluorescein, was designed to solve the well known issues of rapid photobleaching and pH-dependent fluorescent intensity characteristic of the dye fluorescein isothiocyanate.[ citation needed ]
Colour† [5] | Absorb (nm) [6] | Emit (nm) [6] | MM (g/mol)[ citation needed ] | ε (cm−1M−1) [6] | Quantum yield [7] | |
---|---|---|---|---|---|---|
Alexa Fluor 350 | Blue | 346 | 442 | 410 | 19,000 | - |
— 405 | Blue | 401 | 421 | 1028 | 35,000 | - |
— 430 | Green | 434 | 541 | 702 | 15,000 | - |
— 488 | Green | 495 | 519 | 643 | 73,000 | 0.92 |
— 500 | Green | 502 | 525 | 700 | 71,000 | - |
— 514 | Green | 517 | 542 | 714 | 80,000 | - |
— 532 | Yellow | 532 | 554 | 721 | 81,000 | 0.61 |
— 546 | Yellow | 556 | 573 | 1079 | 112,000 | 0.79 |
— 555 | Orange | 555 | 565 | ~1250 | 155,000 | 0.1 |
— 568 | Orange | 578 | 603 | 792 | 88,000 | 0.69 |
— 594 | Red | 590 | 617 | 820 | 92,000 | 0.66 |
— 610 | Red | 612 | 628 | 1172 | 144,000 | - |
— 633 | Far-red | 632 | 647 | ~1200 [8] | 159,000 | - |
— 635 | Far-red | 633 | 647 | - | 140,000 | - |
— 647 | Far-red | 650 | 665 | 1155.06 [9] | 270,000 | 0.33 |
— 660 | Near-IR | 663 | 690 | ~1100 | 132,000 | 0.37 |
— 680 | Near-IR | 679 | 702 | ~1150 | 183,000 | 0.36 |
— 700 | Near-IR | 702 | 723 | ~1400 | 205,000 | 0.25 |
— 750 | Near-IR | 749 | 775 | ~1300 | 290,000 | 0.12 |
— 790 | Near-IR | 782 | 805 | ~1750 | 260,000 | - |
† = approximate colour of the emission spectrum ε = extinction coefficient |
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The Alexa Fluor series dyes are less pH-sensitive and more photostable than the original dyes (fluorescein, rhodamine, etc.) from which they were synthesized.[ citation needed ] While extinction coefficients of each member of this line of dyes are known (see table), quantum yields[ clarification needed ] and life times [ clarification needed ] are not.[ citation needed ] Brightness comparisons are also generally favorable.[ clarification needed ][ citation needed ]
Other commercial product lines provide alternatives to individual members of the line of Alexa Fluor Dyes.[ citation needed ] Comparisons with other dyes are less consistent,[ clarification needed ] and also even more "delicate",[ clarification needed ] depending on the conditions and techniques used.[ editorializing ][ citation needed ] Such comparisons should be considered, depending on the conditions and techniques used, and the dye performance (signal, background, stability) needed.[ editorializing ][ citation needed ]
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 as a fluorescent tracer for many applications.
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.
Rhodamine is a family of related dyes, a subset of the triarylmethane dyes. They are derivatives of xanthene. Important members of the rhodamine family are rhodamine 6G, rhodamine 123, and rhodamine B. They are mainly used to dye paper and inks, but they lack the lightfastness for fabric dyeing.
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.
RiboGreen is a proprietary fluorescent dye that is used in the detection and quantification of nucleic acids, including both RNA and DNA. It is synthesized and marketed by Molecular Probes/Invitrogen of Eugene, Oregon, United States. In its free form, RiboGreen exhibits little fluorescence and possesses a negligible absorbance signature. When bound to nucleic acids, the dye fluoresces with an intensity that, according to the manufacturer, is several orders of magnitude greater than the unbound form. The fluorescence can be detected by a sensor and the nucleic acid can be quantified. The presence of protein contaminants in the sample of nucleic acids to be tested does not make significant contributions to the absorbance, and thus allows for the addition of deoxyribonucleases to the protocol in order to degrade DNA, in the instances where one is only interested in detecting or quantifying RNA.
Invitrogen is one of several brands under the Thermo Fisher Scientific corporation. The product line includes various subbrands of biotechnology products, such as machines and consumables for polymerase chain reaction, reverse transcription, cloning, culturing, stem cell production, cell therapy, regenerative medicine, immunotherapy, transfection, DNA/RNA purification, diagnostic tests, antibodies, and immunoassays.
Fluorescein isothiocyanate (FITC) is a derivative of fluorescein used in wide-ranging applications including flow cytometry. First described in 1942, FITC is the original fluorescein molecule functionalized with an isothiocyanate reactive group (−N=C=S), replacing a hydrogen atom on the bottom ring of the structure. It is typically available as a mixture of isomers, fluorescein 5-isothiocyanate (5-FITC) and fluorescein 6-isothiocyanate (6-FITC). FITC is reactive towards nucleophiles including amine and sulfhydryl groups on proteins. It was synthesized by Robert Seiwald and Joseph Burckhalter in 1958.
Texas Red or sulforhodamine 101 acid chloride is a red fluorescent dye, used in histology for staining cell specimens, for sorting cells with fluorescent-activated cell sorting machines, in fluorescence microscopy applications, and in immunohistochemistry. Texas Red fluoresces at about 615 nm, and the peak of its absorption spectrum is at 589 nm. The powder is dark purple. Solutions can be excited by a dye laser tuned to 595-605 nm, or less efficiently a krypton laser at 567 nm. The absorption extinction coefficient at 596 nm is about 85,000 M−1cm−1.
IAEDANS is an organic fluorophore. It stands for 5-({2-[ amino]ethyl}amino)naphthalene-1-sulfonic acid. It is widely used as a marker in fluorescence spectroscopy.
Rhodamine B is a chemical compound and a dye. It is often used as a tracer dye within water to determine the rate and direction of flow and transport. Rhodamine dyes fluoresce and can thus be detected easily and inexpensively with fluorometers.
Cyanines, also referred to as tetramethylindo(di)-carbocyanines are a synthetic dye family belonging to the polymethine group. Although the name derives etymologically from terms for shades of blue, the cyanine family covers the electromagnetic spectrum from near IR to UV.
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.
In chemistry, a dark quencher is a substance that absorbs excitation energy from a fluorophore and dissipates the energy as heat; while a typical (fluorescent) quencher re-emits much of this energy as light. Dark quenchers are used in molecular biology in conjunction with fluorophores. When the two are close together, such as in a molecule or protein, the fluorophore's emission is suppressed. This effect can be used to study molecular geometry and motion.
The DyLight Fluor family of fluorescent dyes are produced by Dyomics in collaboration with Thermo Fisher Scientific. DyLight dyes are typically used in biotechnology and research applications as biomolecule, cell and tissue labels for fluorescence microscopy, cell biology or molecular biology.
Molecular Probes was a biotechnology company located in Eugene, Oregon specializing in fluorescence. The company was founded in 1975 by Richard and Rosaria Haugland in their kitchen in Minnesota, then moved briefly to Texas and finally to Oregon in the early 1980s.
Lucifer yellow is a fluorescent dye used in cell biology. The key property of Lucifer yellow is that it can be readily visualized in both living and fixed cells using a fluorescence microscope. Lucifer yellow was invented by Walter W. Stewart at the National Institutes of Health and patented in 1978.
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.
The FluoProbes series of fluorescent dyes were developed by Interchim to improve performances of standard fluorophores. They are designed for labeling biomolecules, cells, tissues or beads in advanced fluorescent detection techniques.
Primary and secondary antibodies are two groups of antibodies that are classified based on whether they bind to antigens or proteins directly or target another (primary) antibody that, in turn, is bound to an antigen or protein.
Richard Paul Haugland was an American scientist noted for his work in researching and commercializing fluorescent dyes. He completed his PhD at Stanford in 1970 under Lubert Stryer, showing in a now widely cited and classic paper that Förster resonance energy transfer (FRET) can be used as a "spectroscopic ruler" to measure distances in macromolecules. Haugland founded Molecular Probes in 1975 and continued as its president after the corporation was bought by Invitrogen in 2003. He is the original author of the authoritative volume on molecular probes, The Molecular Probes Handbook, now in its 11th edition.
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has generic name (help) See also Huggett, Brady (30 July 2003). "Invitrogen Prices $325M Worth Of Notes, Keeps Buying Power" . BioWorld.com. Retrieved 1 March 2020.