Many of the classical fluorescent probes that have been successfully utilized for many years in widefield fluorescence, including fluorescein isothiocyanate, Lissamine rhodamine, and Texas red, are also useful in confocal microscopy. Fluorescein is one of the most popular fluorochromes ever designed, and has enjoyed extensive application in immunofluorescence labeling. This xanthene dye has an absorption maximum at 495 nanometers, which coincides quite well with the 488 nanometer (blue) spectral line produced by argon-ion and krypton-argon lasers, as well as the 436 and 467 principal lines of the mercury and xenon arc-discharge lamps (respectively). In addition, the quantum yield of fluorescein is very high and a significant amount of information has been gathered on the characteristics of this dye with respect to the physical and chemical properties. On the negative side, the fluorescence emission intensity of fluorescein is heavily influenced by environmental factors (such as pH), and the relatively broad emission spectrum often overlaps with those of other fluorophores in dual and triple labeling experiments.

The adherent LLC-PK1 cell culture illustrated above was treated with the traditional triple-fluorophore combination of MitoTracker Red CMXRos, Alexa Fluor 488 conjugated to phalloidin and Hoechst 33258. These probes label the mitochondria, filamentous actin network, and DNA in the nucleus, respectively. 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.