Alexa Fluor

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.

History

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]

Dyes and chemistry

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

Comparison with other dyes

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]

Specific comparison studies

Further reading

  • "Molecular Imaging and Contrast Agent Database (MICAD), 2004-2013". Bethesda, MD: National Center for Biotechnology Information (US). 2013. PMID 20641179. Retrieved 1 March 2020. Database entries written by independent experts, on Alexa Fluor product numbers 488, 647, 680, 700, and 750, as well as other conjugated forms.
  • Hullin-Matsuda, F.; Murate, M.; Kobayashi, T. (25 September 2018). "Protein Probes to Visualize Sphingomyelin and Ceramide Phosphoethanolamine". Chem. Phys. Lipids. 216: 132–141. doi:10.1016/j.chemphyslip.2018.09.002. PMID 30194925. S2CID 52179117. Review of a lipid visualization application of the Alexa Fluors.
  • Kozma, E.; Jayasekara, P.S.; Squarcialupi, L.; Paoletta, S.; Moro, S.; Federico, S.; Spalluto, G.; Jacobson, K.A. (1 January 2013). "Fluorescent Ligands for Adenosine Receptors". Bioorg. Med. Chem. Lett. 23 (1): 26–36. doi:10.1016/j.bmcl.2012.10.112. PMC 3557833. PMID 23200243. Review of a set of fluors based on Alexa Fluor (AF) 488, for receptor binding studies. Article presents structures of AF488 and AF532, as well as a large set of other standard flurophores Cy5, Texas Red, EVOBlue, FITC, NBD, Dansyl, 1-Pyrene, and various Bodipy complexes.

References

  1. ^ Invitrogen Staff (6 June 2007). "Alexa Fluor Dyes Spanning the Visible and Infrared Spectrum". Probes.Invitrogen.com. Archived from the original on 11 August 2007. Retrieved 13 August 2007.
  2. ^ ThermoFisher Staff (26 May 2017). "Alexa Fluor Secondary Antibodies". ThermoFisher.com. Retrieved 26 May 2017.
  3. ^ Invitrogen Staff (6 April 2006). "The Alexa Fluor Dye Series". Probes.Invitrogen.com. Archived from the original on 15 August 2007. Retrieved 13 August 2007.
  4. ^ "Invitrogen Prices $325M Worth Of Notes, Keeps Buying Power [uncredited reproduction of 30 July 2003 BioWOrld article]". AllBusiness.com. 4 November 2009. Archived from the original on 24 August 2010. Retrieved 1 March 2020. See also Huggett, Brady (30 July 2003). "Invitrogen Prices $325M Worth Of Notes, Keeps Buying Power". BioWorld.com. Retrieved 1 March 2020.
  5. ^ ThermoFisher Staff (26 February 2020). "Fluorophore Selection". Thermofisher.com. Retrieved 1 March 2020.
  6. ^ a b c LifeTechnologies Staff (26 February 2020). "The Alexa Fluor Dye Series—Note 1.1". LifeTechnologies.com. Retrieved 1 March 2020.
  7. ^ Invitrogen Staff (26 April 2011). "Fluorescence Quantum Yields (QY) and Lifetimes (τ) for Alexa Fluor dyes [Table 1.5]". Invitrogen.com. Retrieved 26 April 2011.
  8. ^ ThermoFisher Staff (8 January 2018). "Alexa Fluor 633 NHS Ester (Succinimidyl Ester)". ThermoFisher.com. Retrieved 8 January 2018.
  9. ^ Esteban, A.; Popp, M.W.; Vyas, V.K.; Strijbis, K.; Ploegh, H.L.; Fink, G.R. (23 August 2011). "Fungal Recognition is Mediated by the Association of Dectin-1 and Galectin-3 in Macrophages". Proc. Natl. Acad. Sci. U.S.A. 108 (34): 14270–14275. Bibcode:2011PNAS..10814270E. doi:10.1073/pnas.1111415108. PMC 3161568. PMID 21825168.
  10. ^ Ballard JL; Peeva VK; deSilva CJ; Lynch JL; Swanson NR (July 2007). "Comparison of Alexa Fluor and CyDye for practical DNA microarray use". Molecular Biotechnology. 36 (3): 175–83. doi:10.1007/s12033-007-0006-4. PMID 17873405. S2CID 37365037. Retrieved 23 October 2010.

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