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.
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 nm).
History
Richard and Rosaria Haugland, the founders of Molecular Probes, are well known in biology and chemistry for their research into fluorescent dyes to be used in biological applications. At the time that Molecular Probes was founded, these products were essentially unavailable commercially. A number of now well-known fluorescent dyes came out of the labs at Molecular Probes. Dyes such as Texas Red, Cascade Blue, Oregon Green, Marina Blue and the Alexa Fluor family. Arguably the most famous, the Alexa Fluor family of dyes, were intended to improve upon the properties of previously developed biological fluorescent dye families and solve some of the issues that they possessed. Structurally, the Alexa Fluor dyes are generated through the sulfonation and additional modification of certain well known dye families. In particular, the dye families of coumarin, rhodamine, xanthene (of which the industry-standard fluorescein is a member), and cyanine dyes were used. Sulfonation makes the Alexa Fluor dyes negatively charged and more hydrophilic than their precursors, while additional modification was used to improve the performance in other areas. For example, Alexa Fluor 488, a sulfonated and chemically modified form of fluorescein, was designed to solve the well known issues of rapid photo-bleaching and pH dependent fluorescent intensity common in the industry-standard FITC dye. The Alexa Fluor dyes were named after Alex Haugland, son of Richard and Rosaria Haugland. Molecular Probes was acquired in 2003, by Invitrogen[4] who further worked to expand the Alexa Fluor family adding new dyes to fill gaps in the spectrum. In 2008, Invitrogen became Life Technologies when they merged with Applied Biosystems, bringing the Alexa Fluor family of dyes under the new Life Technologies name. In 2014, Life Technologies was acquired by Thermo Fisher Scientific who revitalized the Invitrogen name and brought the Alexa Fluor family of dyes back under the Invitrogen brand.
Dyes
| Colour†[citation needed] | Absorb (nm)[5] |
Emit (nm)[5] |
MM (g/mol)[citation needed] |
ε (cm−1M−1)[5] |
Quantum Yield [6] | |
|---|---|---|---|---|---|---|
| Alexa Fluor 350 | blue | 346 | 442 | 410 | 19,000 | - |
| — 405 | violet | 401 | 421 | 1028 | 35,000 | - |
| — 430 | green | 434 | 541 | 702 | 15,000 | - |
| — 488 | cyan-green | 495 | 519 | 643 | 73,000 | 0.92 |
| — 500 | green | 502 | 525 | 700 | 71,000 | - |
| — 514 | green | 517 | 542 | 714 | 80,000 | - |
| — 532 | green | 532 | 554 | 721 | 81,000 | 0.61 |
| — 546 | yellow | 556 | 573 | 1079 | 112,000 | 0.79 |
| — 555 | yellow-green | 555 | 565 | ~1250 | 155,000 | 0.1 |
| — 568 | orange | 578 | 603 | 792 | 88,000 | 0.69 |
| — 594 | orange-red | 590 | 617 | 820 | 92,000 | 0.66 |
| — 610 | red | 612 | 628 | 1172 | 144,000 | - |
| — 633 | Far-red | 632 | 647 | ~1200[7] | 159,000 | - |
| — 635 | Far-red | 633 | 647 | - | 140,000 | - |
| — 647 | Far-red | 650 | 665 | 1155.06[8] | 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 color of the emission spectrum ε = extinction coefficient | ||||||
Comparison with other dyes
While extinction coefficients are known (see the table above), quantum yields and life times are not. Comparisons with other dyes should be considered depending on the conditions (technique) used and performance (signal, background, stability) needed.
The Alexa Fluor series dyes are less pH-sensitive and more photostable than the original dyes (fluorescein, rhodamine, etc.) from which they were synthesized. Brightness comparisons are also generally favorable. Comparisons with other dyes are less consistent, and also even more delicate, depending on the conditions (technique) used. A third party has compared Alexa Fluor 647 dye with Cy5 (similar wavelength), conjugated to DNA.[9]
Similar lines of fluorescent dyes provide an alternative to the Alexa Fluor Dyes (see also the list in Category:Fluorescent dyes).
References
- ^ "Alexa Fluor Dyes Spanning the Visible and Infrared Spectrum". 6 June 2007. Archived from the original on 11 August 2007. Retrieved 13 August 2007.
{{cite web}}: Unknown parameter|deadurl=ignored (|url-status=suggested) (help) - ^ "Alexa Fluor Secondary Antibodies". www.thermofisher.com. Retrieved 26 May 2017.
- ^ "The Alexa Fluor Dye Series". Molecular Probes, Inc. 6 April 2006. Archived from the original on 15 August 2007. Retrieved 13 August 2007.
{{cite web}}: Unknown parameter|deadurl=ignored (|url-status=suggested) (help) - ^ "Allbusiness.com: Invitrogen Prices $325M Worth Of Notes, Keeps Buying Power". Retrieved 17 January 2010.
- ^ a b c http://www.lifetechnologies.com/sg/en/home/references/molecular-probes-the-handbook/technical-notes-and-product-highlights/the-alexa-fluor-dye-series.html
- ^ "Fluorescence quantum yields (QY) and lifetimes (τ) for Alexa Fluor dyes—Table 1.5". Retrieved 26 April 2011.
- ^ "Alexa Fluor™ 633 NHS Ester (Succinimidyl Ester)". Retrieved 8 January 2018.
- ^ Supporting Information; Esteban, Fink, et al. Fungal recognition is mediated by the association of dectin-1 and galectin-3 in macrophages. Proceedings of the National Academy of Sciences of the United States of America. 2011. Vol 108, no 34, pg 14270-14275. 10.1073/pnas.1111415108
- ^ Ballard JL; Peeva VK; deSilva CJ; Lynch JL; Swanson NR (July 2007). "Comparison of Alexa Fluor and CyDye for practical DNA microarray use". Retrieved 23 October 2010.