A triple fluorophore combination of MitoTracker Orange CMTMRos, BODIPY FL conjugated to phallacidin, and TO-PRO-3 was used to label an adherent culture of human brain glioma cells for mitochondria, the F-actin network, and nuclear DNA, respectively. The cells were first treated with the MitoTracker probe in growth medium for one hour, washed and fixed with paraformaldehyde (prepared in growth medium), permeabilized, and blocked with bovine serum albumen. The cells were subsequently labeled with the conjugated phallotoxin and counterstained with the cyanine monomer (TO-PRO-3) reagent. Images were recorded with a 60x oil immersion objective using a zoom factor of 1.5 and sequential scanning with the 488-nanometer spectral line of an argon-ion laser, the 543-nanometer line from a green helium-neon laser, and the 633-nanometer line of a red helium-neon laser. During the processing stage, the MitoTracker Orange CMTMRos image channel was pseudocolored yellow, while the BODIPY FL image channel was pseudocolored green and the TO-PRO-3 channel was pseudocolored red.