Fluorescent probe technology and cell biology were dramatically altered by the discovery of the green fluorescent protein (GFP) from jellyfish and the development of mutant spectral variants, which have opened the door to non-invasive fluorescence multicolor investigations of subcellular protein localization, intermolecular interactions, and trafficking using living cell cultures. More recently, the development of nanometer-sized fluorescent semiconductor quantum dots has provided a new avenue for research in confocal and widefield fluorescence microscopy. Despite the numerous advances made in fluorescent dye synthesis during the past few decades, there is very little solid evidence about molecular design rules for developing new fluorochromes, particularly with regard to matching absorption spectra to available confocal laser excitation wavelengths. As a result, the number of fluorophores that have found widespread use in confocal and fluorescence microscopy is a limited subset of the many thousands that have been discovered.

The adherent culture of U-2 OS cells presented in the digital image above was transfected with a pEYFP-Mitochondria plasmid subcellular localization vector, which contains the mitochondrial targeting sequence from subunit VIII of human cytochrome C oxidase. The enhanced yellow fluorescent protein gene employed with this culture features several important amino acid substitutions that shift the emission maximum of green fluorescent protein (GFP) by approximately 18 nanometers, from 509 to 527 nanometers. The cells were additionally labeled with Alexa Fluor 568 conjugated to phalloidin and Hoechst 33258, targeting the filamentous actin network and nuclei, 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.