Mitochondrial probes are among the most useful fluorophores for investigating cellular respiration and are often employed along with other dyes in multiple labeling investigations, such as the combination employed for the image presented in this section. The traditional probes, rhodamine 123 and tetramethylrosamine, are rapidly lost when cells are fixed and have largely been supplanted by newer, more specific, fluorophores developed by Molecular Probes. These include the popular MitoTracker and MitoFluor series of structurally diverse xanthene, benzoxazole, indole, and benzimidazole heterocycles that are available in a variety of excitation and emission spectral profiles. The mechanism of action varies for each of the probes in this series, ranging from covalent attachment to oxidation within respiring mitochondrial membranes.

The Swiss mouse embryo fibroblast cell culture illustrated above was triple-labeled with the standard protocol: MitoTracker Red CMXRos to target the mitochondria, Alexa Fluor 488 conjugated to phalloidin for the filamentous actin network, and Hoechst 33342 to stain DNA in the cell nuclei. 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.