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DOI: 10.1369/jhc.4A6355.2005
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Journal of Histochemistry and Cytochemistry
Volume 53 (8): 941-953, 2005
Copyright ©The Histochemical Society, Inc.

New Methods to Evaluate Colocalization of Fluorophores in Immunocytochemical Preparations as Exemplified by a Study on A2A and D2 Receptors in Chinese Hamster Ovary Cells

Luigi F. Agnati, Kjell Fuxe, Maria Torvinen, Susanna Genedani, Rafael Franco, Stan Watson, Gastone G. Nussdorfer, Giuseppina Leo and Diego Guidolin

Department of Biomedical Sciences, Section of Physiology (LFA,GL) and Section of Pharmacology (SG), University of Modena, Modena, Italy; Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden (KF,MT); Department of Biochemistry and Molecular Biology, University of Barcelona, Barcelona, Spain (RF); Mental Health Research Institute, University of Michigan, Ann Arbor, Michigan (SW); and Department of Human Anatomy and Physiology, Section of Anatomy, University of Padova, Padova, Italy (GGN,DG)

Correspondence to: Diego Guidolin, Department of Human Anatomy and Physiology, Section of Anatomy, University of Padova, via Gabelli 65, 35121 Padova, Italy. E-mail: diego.guidolin{at}unipd.it

An important aspect of the image analysis of immunocytochemical preparations is the evaluation of colocalization of different molecules. The aim of the present study is to introduce image analysis methods to identify double-labeled locations exhibiting the highest association of two fluorophores and to characterize their pattern of distribution. These methods will be applied to the analysis of the cotrafficking of adenosine A2A and dopamine D2 receptors belonging to the G protein–coupled receptor family and visualized by means of fluorescence immunocytochemistry in Chinese hamster ovary cells after agonist treatment. The present procedures for colocalization have the great advantage that they are, to a large extent, insensitive to the need for a balanced staining with the two fluorophores. Thus, these procedures involve image processing, visualization, and analysis of colocalized events, using a covariance method and a multiply method and the evaluation of the identified colocalization patterns. Moreover, the covariance method offers the possibility of detecting and quantitatively characterizing anticorrelated patterns of intensities, whereas the immediate detection of colocalized clusters with a high concentration of labeling is a possibility offered by the multiply method. The present methods offer a new and sensitive approach to detecting and quantitatively characterizing strongly associated fluorescence events, such as those generated by receptor–receptor interaction, and their distribution patterns in dual-color confocal laser microscopy.

(J Histochem Cytochem 53:941–953, 2005)

Key Words: clusters • colocalization • computer-assisted analysis • confocal laser microscopy • receptor


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