Marketed by a number of distributors, the cyanine dyes are readily available as reactive dyes or fluorophores coupled to a wide variety of secondary antibodies, dextrin, streptavidin, and egg-white avidin. The cyanine dyes generally have broader absorption spectral regions than members of the Alexa Fluor family, making them somewhat more versatile in the choice of laser excitation sources for confocal and fluorescence microscopy. For example, using the 547-nanometer spectral line from an argon-ion laser, Cy2 is about twice as efficient in fluorescence emission as Alexa Fluor 488. In an analogous manner, the 514-nanometer argon-ion laser line excites Cy3 with a much higher efficiency than Alexa Fluor 546, a spectrally similar probe. Emission profiles of the cyanine dyes are comparable in spectral width to the Alexa Fluor series.

To produce the digital image featured above, a fixed and permeabilized adherent culture of Madin-Darby canine kidney cells was immunofluorescently labeled with primary mouse anti-cytokeratin antibodies, followed by goat anti-mouse Fab fragments conjugated to the cyanine dye, Cy2. Before fixation, the culture was treated for one hour with MitoTracker Red CMXRos, and after the antibody treatments, the nuclei were counterstained with Hoechst 33258. Images were recorded in grayscale with a QImaging Retiga Fast-EXi camera system coupled to an Olympus BX-51 microscope equipped with bandpass emission fluorescence filter optical blocks provided by Omega Optical. During the processing stage, individual image channels were pseudocolored with RGB values corresponding to each of the fluorophore emission spectral profiles.