Mitochondrial probes are among the most useful fluorophores for investigating cellular respiration and are often employed along with other dyes in multiple labeling investigations. 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.

In order to generate the digital image presented above, an adherent MDOK cell culture in log phase growth was treated with MitoTracker Red CMXRos for one hour before being fixed and permeabilized. The cells were subsequently blocked with bovine serum albumen and incubated with Alexa Fluor 350 conjugated to phalloidin, followed by counterstaining with SYTOX Green. 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.