Localization of specific peptides, proteins, and macromolecular complexes to the mitochondria in mammalian cells is generally accomplished through the use of peptide signals that mediate transport to the organelle. Recombinant plasmids have been constructed that contain a fusion protein consisting of the yellow-green variant (referred to as enhanced yellow fluorescent protein; EYFP) of the Aequorea victoria green fluorescent protein (GFP) coupled to the mitochondrial targeting nucleotide sequence from subunit VIII of human cytochrome C oxidase. Upon transcription and translation of the plasmid in transfected mammalian hosts, the mitochondrial localization signal is responsible for transport and distribution of the fluorescent protein chimera throughout the cellular mitochondrial network. Tubular mitochondria can be subsequently visualized using fluorescence microscopy.

The culture of Madin-Darby canine kidney cells presented in the digital image above was labeled with DAPI and Alexa Fluor 568 conjugated to phalloidin, which target DNA in the cell nucleus and the F-actin cytoskeletal network, respectively. In addition, the cells were transfected with a pEYFP-Mitochondria (enhanced yellow fluorescent protein) chimeric plasmid subcellular localization vector. 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.