Automated method for quantifying fluorophore colocalization in fluorescence double-labeling experiments.

Fluorescence double-labeling is an important method for investigating associations of molecules (1). For example, evidence of co-association of proteins often includes demonstrating that they are colocalized in the same compartment of the cell. Furthermore, colocalization studies are not limited to proteins but also include measuring the colocalization of separate lipid species (8), lipids with proteins (9), and nucleic acids (4,5). Thus, double-labeling experiments are performed to address a broad range of questions. Quantification of fluorophore colocalization is an important feature of double-labeling experiments, and methods for accomplishing this have been described (12). One important method for quantifying fluorophore colocalization is that of the cross-correlation analysis (13). Several recent studies have provided important examples of using this type of analysis to quantitate protein associations in cells (6,7,10,11,13). Cross-correlation analysis is a multi-step process that has been difficult to accomplish for large numbers of cells. To address this issue, a method for quantifying fluorophore colocalization in a facile manner using the cross-correlation analysis was written using scriptable imaging software. With this tool, quantitation of fluorophore colocalization is largely automated, thus allowing for fast and convenient quantitation of large numbers of cells. In the cross-correlation analysis, fluorophore colocalization is measured by determining the correlation coefficient (ρ) for the separate fluorophores using the equation: