The number of fluorescent probes currently available for confocal and fluorescence microscopy runs in the hundreds, with many dyes having absorption maxima closely associated with common laser spectral lines. An exact match between a particular laser line and the absorption maximum of a specific probe is not always possible, but the excitation efficiency of lines near the maximum is usually sufficient to produce a level of fluorescence emission that can be readily detected. Consider, for example, fluorescein isothiocyanate (FITC), which has an absorption maximum of 495 nanometers. Excitation of the FITC fluorophore at 488 nanometers using an argon-ion laser produces an emission efficiency of approximately 87 percent. In contrast, when the 477-nanometer or the 514-nanometer argon-ion laser lines are used to excite FITC, the emission efficiency drops to only 58 or 28 percent, respectively. Clearly, the 488-nanometer argon-ion (or krypton-argon) laser line is the most efficient source for excitation of this fluorophore.

The single cell featured in the digital image above was resident in a COS-7 culture immunofluorescently labeled with primary anti-tubulin mouse monoclonal antibodies followed by goat anti-mouse Fab fragments conjugated to fluorescein isothiocyanate (FITC), which has an absorption maximum of 495 nanometers. The culture was counterstained for DNA in the cell nucleus with the ultraviolet-absorbing probe DAPI. 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.