New methods to evaluate colocalization of fluorophores in immunocytochemical preparations as exemplified by a study on A(2A) and D-2 receptors in Chinese hamster ovary cells

An important aspect in the image analysis of immunocytochemical preparations is the evaluation of co-localization of different molecules. The aim of the present study is to introduce image analysis methods to identify double-labelled locations exhibiting the highest association of two fluorophores and to characterize their pattern of distribution. They will be applied to the analysis of the co-trafficking of Adenosine A2A and Dopamine D2 receptors belonging to the G-protein coupled receptor (GPCR) family and visualized by means of fluorescence immunocytochemistry (ICC) in CHO cells after agonist treatment. The present procedures for co-localization have the great advantage that they are to a large extent insensitive to the need of a balanced staining with the two fluorophores. Thus, these procedures involve image processing, visualization and analysis of co-localized events, using a covariance method and a multiply method and the evaluation of the identified co-localization patterns. Moreover, the covariance method offers the possibility to detect and quantitatively characterize anti-correlated patterns of intensities, while the immediate detection of co-localized clusters with high concentration of labelling is a possibility offered by the multiply method. The present methods offer a new and sensitive approach to detect and quantitatively characterize strongly associated fluorescence events, such as those generated by receptor-receptor interaction, and their distribution patterns in dual-colour confocal laser microscopy.