Alexa Fluor dyes are often used as labels for cells and tissues in fluorescence microscopy and cell biology. Alexa Fluor dyes can be conjugated directly to primary or secondary antibodies to amplify signal and sensitivity or other biomolecules. The excitation and emission spectra of the Alexa Fluor series cover the visible spectrum and extend to the infrared. Individual members of this family are roughly numbered according to their maximum excitation (in nm).
Structurally, Alexa Fluor dyes are generated by sulfonation and additional modifications to certain well-known dye families. In particular, a family of dyes using coumarin, rhodamine, x-tons (of which industry standard fluorescein is a member) and cyanine dyes are used. Sulfonation makes Alexa Fluor dyes negatively charged and more hydrophilic than their precursors, while other modifications have been made to improve performance in other areas. For example, Alexa Fluor 488 is a sulfonated and chemically modified fluorescein designed to solve the well-known problems of fast photobleaching and pH-dependent fluorescence intensity common to industry standard FITC dyes.
Although the extinction coefficient is known, the quantum yield and lifetime are unknown. Comparison with other dyes should be considered based on the conditions (technology) used and the required performance (signal, background, stability). Compared with the original dyes that synthesized them (fluorescein, rhodamine, etc.), Alexa Fluor series dyes are less sensitive to pH and have higher light stability. Brightness comparisons are usually also advantageous. Depending on the conditions (techniques) used, the comparison with other dyes is inconsistent and even finer. Third parties have compared Alexa Fluor 647 dye to Cy5 (similar wavelength) coupled to DNA. A similar line of fluorescent dyes offers alternatives to Alexa fluorescent dyes.