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What is Alexa Fluor? How it compare with others?

Excitation and emission spectra of the Alexa Fluor series cover the visible spectrum range and extend into the infrared. The numbering of the individual family members correlates roughly with their excitation maxima (in nm).

Sulfonation of coumarin, rhodamine, xanthene (such as fluorescein), and cyanine dyes allows the synthesis of Alexa Fluor dyes. Sulfonation makes Alexa Fluor dyes negatively charged and hydrophilic. Alexa Fluor dyes are generally more stable, brighter, and less pH-sensitive than common dyes (e.g. fluorescein, rhodamine) of comparable excitation and emission, and to some extent the newer cyanine series. However, they are also more expensive. They are patented by Invitrogen (which acquired the company that developed the Alexa dyes, Molecular Probes) and thus are priced higher than the common dyes that are available from multiple manufacturers.


Similar alternatives include the 
DyLight Fluors from Pierce (Thermo Fisher Scientific), and the Atto series from Atto-Tec and sold by Sigma-Aldrich.

 

Alexa Fluor 350 blue 346 442 410 19,000
— 405 violet 401 421 1028 34,000
— 430 green 434 541 702 16,000
— 488 green 495 519 643 71,000
— 500 green 502 525 700 71,000
— 514 green 517 542 714 80,000
— 532 green 532 554 721 81,000
— 546 yellow-green 556 573 1079 104,000
— 555 green 555 565 ~1250 150,000
— 568 orange 578 603 792 91,300
— 594 orange-red 590 617 820 90,000
— 610 red 612 628 1172 138,000
— 633 not vis 632 647 ~1200 100,000
— 647 not vis 650 665 ~1300 239,000
— 660 not vis 663 690 ~1100 132,000
— 680 not vis 679 702 ~1150 184,000
— 700 not vis 702 723 ~1400 192,000
— 750 not vis 749 775 ~1300 240,000
?=approximate color of the emission spectrum; e=extinction coefficient


Comparison with other dyes

The Alexa series dyes are less pH-sensitive and more photostable than the original dyes (fluorescein, rhodamine, etc.) from which they were synthesized. However, brightness comparisons are not presently available. Brightness is commonly measured as a product of extinction coefficient (absorption efficiency) and quantum yield (emission efficiency) [5] While extinction coefficients are known (see the table above), the quantum yields of the Alexas have not been published by Molecular Probes.

In one case, a third party has compared one Alexa with another commonly used dye. This was a comparison of Cy5 and its Alexa with similar wavelength, Alexa 647, with the dyes conjugated to DNA.[1] This study found that Cy5 is brighter, but less photostable than Alexa 647.


Therefore presently it is difficult to quantitatively choose the best dye for a particular application and in many cases empirical testing is in order.

Reference

(1) Alexa Fluor Dyes - Across the Spectrum.

(2) The Alexa Fluor Dye Series. biotium, ThermoFisher

(3) Panchuk-Voloshina N, Haugland RP, Bishop-Stewart J, et al (1999). "Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates". J. Histochem. Cytochem. 47 (9): 1179-88. [SAGE journals]

(4) Berlier JE, Rothe A, Buller G, et al (2003). "Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy dyes: fluorescence of the dyes and their bioconjugates". J. Histochem. Cytochem. 51 (12): 1699-712. [SAGE journals]

(5) Souslova EA, Belousov VV, Lock JG, Stromblad S, Kasparov S, Bolshakov AP, Pinelis VG, Labas YA, Lukyanov S, Mayr LM, Chudakov DM (2007). "Single fluorescent protein-based Ca2+ sensors with increased dynamic range". BMC Biotechnol. 7 (7): 37. [PMC]