Alexa Fluor dyes are available in a broad range of fluorescence excitation and emission wavelength maxima, ranging from the ultraviolet and deep blue to the near-infrared regions. Alphanumeric names of the individual dyes are associated with the specific excitation laser or arc-discharge lamp spectral lines for which the probes are intended. For example, Alexa Fluor 488 is designed for excitation by the blue 488-nanometer line of the argon or krypton-argon ion lasers, while Alexa Fluor 568 is matched to the 568-nanometer spectral line of the krypton-argon laser. Several of the Alexa Fluor dyes are specifically designed for excitation by either the blue diode laser (405 nanometers), the orange/yellow helium-neon laser (594 nanometers), or the red helium-neon laser (633 nanometers). Other Alexa Fluor dyes are intended for excitation with traditional mercury arc-discharge lamps in the visible (Alexa Fluor 546) or ultraviolet (Alexa Fluor 350, also useful with high-power argon-ion lasers), and solid-state red diode lasers (Alexa Fluor 680). Because of the large number of available excitation and emission wavelengths in the Alexa Fluor series, multiple labeling experiments can often be conducted exclusively with these dyes.

One of the most useful fluorophore combinations for visualizing internal cellular details includes MitoTracker Red CMXRos to target mitochondria, Alexa Fluor 488 conjugated to phalloidin for the filamentous actin network, and DAPI to localize the nuclei. The cell culture illustrated above was stained with this protocol. 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.