A popular fluorescent protein derivative, the yellow fluorescent protein (YFP) was designed on the basis of the GFP crystalline structural analysis to red-shift the absorption and emission spectra. Yellow fluorescent protein is optimally excited by the 514-nanometer spectral line of the argon-ion laser, and provides more intense emission than enhanced green fluorescent protein, but is more sensitive to low pH and high halogen ion concentrations. The enhanced yellow fluorescent protein derivative (EYFP) is useful with the 514 argon-ion laser line, but can also be excited with relatively high efficiency by the 488-nanometer line from argon and krypton-argon lasers. Both of these fluorescent protein derivatives have been widely applied to protein-protein FRET investigations in combination with CFP, and in addition, have proven useful in studies involving multiprotein trafficking.

The culture of human lung carcinoma cells illustrated 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 used with this culture contains 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 the nucleic acid stain SYTOX Orange and Alexa Fluor 350 conjugated to phalloidin, targeting DNA and filamentous actin, 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.