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]