A popular traditional probe that is useful in confocal and fluorescence microscopy is the phenanthridine derivative, propidium iodide, first synthesized as an anti-trypanosomal agent along with the closely related ethidium bromide. Propidium iodide binds to DNA in a manner similar to the acridines (via intercalation) to produce orange-red fluorescence centered at 617 nanometers. The positively charged fluorophore also has a high affinity for double-stranded RNA. Propidium has an absorption maximum at 536 nanometers, and can be excited by the 488-nanometer or 514-nanometer spectral lines of an argon-ion (or krypton-argon) laser, or the 543-nanometer line from a green helium-neon laser. The dye is often employed as a counterstain to highlight cell nuclei during double or triple labeling of multiple intracellular structures. Environmental factors can affect the fluorescence spectrum of propidium, especially when the dye is used with mounting media containing glycerol. The structurally similar ethidium bromide, which also binds to DNA by intercalation, produces more background staining and is therefore not as effective as propidium.

The culture of transformed African green monkey kidney cells featured in the digital image above was labeled with Alexa Fluor 350 conjugated to wheat germ agglutinin, a fluorescent lectin that selectively binds to sialic acid residues, which are found in both mucoproteins and glycoproteins. The cells were also stained with Alexa Fluor 488 conjugated to phalloidin and the intercalating dye propidium iodide, which target the cytoskeletal filamentous actin network and DNA, 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.