substituents, carboxyl, acetylmethoxy, and sulfo groups which alter their hydrophilicity. Ex (nm): Excitation wavelength in nanometers Em (nm): Emission wavelength in nanometers MW: Molecular weight QY: Quantum yield * Depends strongly on viscosity, temperature, and biomolecular interactions. Common cyanine dyes and their uses Because they yield brighter and more stable fluorescence, cyanines can advantageously replace conventional dyes such as fluorescein and rhodamines. Cy3 and Cy5 are the most popular, used typically combined for 2 colors detection. Cy3 fluoresces greenish yellow (~550 nm excitation, ~570 nm emission), while Cy5 is fluorescent in the far-red region (~650 excitation, 670 nm emission). Cy3 can be detected by various fluorometers, imagers, and microscopes with standard filters for Tetramethylrhodamine (TRITC). Due to its high molar extinction coefficient, this dye is also easily detected by naked eye on electrophoresis gels, and in solution. Cy5 became a popular replacement for far red fluorescent dyes because of its high extinction coefficient (as small as 1 nanomol can be detected in gel electrophoresis by naked eye) and its fluorophore emission maximum in the red region, where many CCD detectors have maximum sensitivity and biological objects give low background interference. The scanners actually use diverse laser emission wavelengths (typically 532 nm and 635 nm) and filter wavelengths (550-600 nm and 655-695 nm) to avoid background contamination. They are thus able to easily distinguish colors from Cy3 and from Cy5, and also able to quantify the amount of Cy3 and Cy5 labeling in one sample (multiparametric detection). Other cyanine dyes are useful: Cy3.5 can replace sulfoRhodamine 101. Cy5.5 is a near-infrared (IR) fluorescence-emitting dye (excitation/emission maximum 678/694 nm). Cy7 is a near-IR fluor that is invisible to the naked eye (excitation/emission maximum 750/776 nm). It is used in in vivo imaging applications, as well as the Cy7.5 dye. Sulfo–Cyanine dyes bear one or two sulfo groups, rendering the Cy dye water-soluble, but tri- and quadri-sulfonated forms are available for even higher water solubility. PEGylation is another modification that confers hydrophilicity, not only to the dye but also to the labeled conjugate. Nomenclature and structure The Cy3 and Cy5 nomenclature was first proposed by Ernst, et al. in 1989, and is non-standard since it gives no hint of their chemical structures. In the original paper the number designated the count of the methines (as shown), and the side chains were unspecified. Due to this ambiguity various structures are designated Cy3 and Cy5