Lucifer Yellow from Invitrogen

Green Fluorescent Protein (GFP) has revolutionized molecular biology by giving researchers the ability to label any protein and track it in fixed or living cells. There are now too many variations of GFP to keep up with, but researchers have adapted GFP to produce different colors (such as Cherry), or to only fluorescence under specific conditions (such as pH sensitive GFP).

At times, researchers need the ability to label cells without specific protein GFP constructs. Examining fluid uptake by cells, vesicle transport within cells, or testing the permeability of monolayers all require small fluorescent compounds. One of the first compounds to fill this role was fluorescein, later fluorescein isothiocyanate (FITC). This green fluorescent molecule is bright and nontoxic to cells, but like most small fluorochromes, it can bleach during imaging. Lucifer Yellow is an alternative to FITC, and is unique due to its high stokes range, of about 110 nanometers. In addition, Lucifer Yellow is highly soluble in water allowing high concentrations of the dye to be used when necessary, which is not possible with the less water soluble FITC. Although Lucifer Yellow can lose intensity during imaging, antibodies for Lucifer Yellow can be used to maintain a better signal during imaging if need be.

Another drawback to these dyes is that they cannot readily label biologically relevant molecules such as proteins or lipids. Covalently linking these dyes to proteins results in a loss of fluorescent intensity and with the characteristic sensitivity to photobleaching, these labeled proteins quickly lose signal during imaging. Alternative labeling approaches must be considered when labeling biomolecules.

In my lab, we use Lucifer Yellow to label endocytic compartments in cells. Due to the high water solubility, bright fluorescence, low cell toxicity, and broad stokes shift, we are able to visualize endocytic compartments in fixed and living cells. Recently, we have also used this dye to determine the tight junctions formed by cells grown in a transwell system. These tight junctions do not allow small molecules to pass from the apical side to the basolateral, and so Lucifer Yellow was restrained from entering the basolateral compartment in the presence of a complete monolayer.

This is an essential tool for many imaging experiments that cannot be performed with the available alternatives.