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 fluorescence microscopy is a limited subset of the many thousands that have been discovered.

After fixation and permeabilization, the culture of MDCK cells presented above was labeled with propidium iodide and Alexa Fluor 350 conjugated to phalloidin, which target DNA and filamentous actin, respectively. In addition, the cells were first transfected with a pEYFP-Mitochondria plasmid subcellular localization vector, thus localizing a yellow fluorescent protein tag to the intracellular mitochondrial network. 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.