With steady improvement of the reliability of immunofluorescence techniques and quality of confocal microscopes, quantitative colocalization analysis of confocal images emerged as a powerful tool capable of closing the gap between anecdotal evidence and the actual proof of existence of colocalization.

Colocalization is defined as the presence of two or more types of fluorescence molecules at the same location. Physically, this means that the colors emitted by them occupy the same pixel in the image. Biologically, this means that two or more different molecules attach to the same structure in the cell. In the context of digital imaging this is described as the spatial overlap of two or more dyes in a multichannel image. Although colocalization is relative, the information on the appearance of distinct molecules at the same location can be of particular significance to researchers. If the question is “Does protein A overlap/colocalize with protein B in a given image, and if so to what degree?”, quantitative colocalization provides the answer. The degree of colocalization in medico-biological specimens can not be judged by naked eye even when seems obvious. Since colocalization is characterized by the change of the color of dyes, reliable estimation of the degree of this change should be performed exclusively by analyzing colocalization coefficients.

It is important to follow several guidelines to have your colocalization images suitable for quantitation. Ignoring them can undermine reliability of quantitative colocalization analysis or even make your confocal images fully unusable for it.

CoLocalizer Pro software estimates colocalization by calculating a number of values representing the proportion of colocalized pixels in dual-color images. These values are called colocalization coefficients.

Coefficients calculated by CoLocalizer Pro and their significance:

Pearson's correlation coefficient is one of the standard measures in pattern recognition. It is used for describing the correlation of the intensity distributions between channels. It takes into consideration only similarity between shapes, while ignoring the intensities of signals:

Overlap coefficient according to Manders indicates an overlap of the signals and thus represents the true degree of colocalization. This coefficient is not sensitive to the limitations of typical fluorescence imaging, such as efficiency of hybridization, sample photobleaching, and camera quantum efficiency:

Overlap coefficients k1 and k2 split the value of colocalization into the two separate parameters. k1 and k2 coefficients depend on the sum of the products of the intensities of two channels. Thus, they are sensitive to the differences in the intensities of signals:

Colocalization coefficients m1 and m2 describe the contribution of each channel to the image ROI. They are not sensitive to the intensities of signals and can be used when the numbers of objects are not equal:

Colocalization coefficients M1 and M2 describe the contribution of each channel to the scatter gram ROI. They are not sensitive to the intensities of signals and can be used when the numbers of objects are not equal:

Colocalization window of CoLocalizer Pro displays Scatter Gram, preview of the Current Image ROI, a pair of channels according to which the calculations are performed, and the results of coefficients calculations. All coefficients calculated by CoLocalizer Pro help to determine the degree of colocalization of antigens, but they estimate colocalization differently. Researchers can choose to calculate one, two, or all of them depending on the specifics of their experiments.

Top-ranked medico-biological journals publish studies performed with the help of CoLocalization Research Software:

2007

Amazit L et al (2007) Regulation of SRC-3 intercompartmental dynamics by estrogen receptor and phosphorylation. Mol Cell Biol 27:6913-6932.

Mahon KP et al (2007) Deconvolution of the cellular oxidative stress response with organelle-specific peptide conjugates. Chem Biol 14:923-930.

Zinchuk V et al (2007) Quantitative colocalization analysis of multicolor confocal immunofluoresscence microscopy images: pushing pixels to exlpore biological phenomena. Acta Histochem Cytochem 40:101-111.

Marinkovic D et al (2007) Foxo3 is required for the regulation of oxidative stress in erythropoiesis. J Clin Invest 117:2133-2144.

Watanabe T et al (2007) Involvement of host cellular multivesicular body functions in hepatitis B virus budding. PNAS 104:10205-10210.

Van Acker GJ et al (2007) Cause-effect relationships between zymogen activation and other early events in secretagogue-induced acute pancreatitis. Am J Physiol Gastrointest Liver Physiol 292:G1738-G1746.

Desplanques AS et al (2007) Tyrosine phosphorylation and lipid raft association of pseudorabies virus glycoprotein E during antibody-mediated capping. Virology 362:60-66.

Zinchuk V et al (2007) Ethanol consumption alters expression and colocalization of bile salt export pump and multidrug resistance protein 2 in the rat. Histochem Cell Biol 127:503-512.

Clizbe D et al (2007) IDI2, a second isopentenyl diphosphate isomerase in mammals. J Biol Chem 282:6668-6676.

Berg KA et al (2007) Integrins regulate opioid receptor signaling in trigeminal ganglion neurons. Neuroscience 144:889-897.

Röcker C et al (2007) Affinity of C-reactive protein toward FcRI is strongly enhanced by the ϒ-chain. Am J Pathol 170:755-763.

Mutch CM et al (2007) Activation-induced endocytosis of the raft-associated transmembrane adaptor protein LAB/NTAL in B lymphocytes: evidence for a role in internalization of the B cell receptor. Int Immunol 19:19-30.

2006

Rey O et al (2006) Requirement of the TRPC1 cation channel in the generation of transient Ca2+ oscillations by the calcium-sensing receptor. J Biol Chem 281:38730-38737.

Head BP et al (2006) Microtubules and actin microfilaments regulate lipid raft/caveolae localization of adenylyl cyclase signaling components. J Biol Chem 281:26391-26399.

Zinchuk O and Zinchuk V (2006) Dynamics of cellular responses studied by quantitative colocalization analysis. Microscopy and Analysis 20:S13-S15.

Tsutsumi YM et al (2006) Role of 12-lipoxygenase in volatile anesthetic-induced delayed preconditioning in mice. Am J Physiol Heart Circ Physiol 291:H979-983.

Swaney JS et al (2006) Focal adhesion in (myo)fibroblasts scaffold adenylyl cyclase with phosphorylated caveolin. J Biol Chem 281:17173-17179.

Patel HH et al (2006) Protection of adult cardiomyocytes from ischemic cell death: role of caveolar microdomains and δ-opioid receptors. Am J Physiol Heart Circ Physiol 291:H344-H350.

Cario E et al (2006) Trypsin-sensitive modulation of intestinal epithelial MD-2 as mechanism of lipopolysaccharide tolerance. J Immunol 176:4258-4266.

2005

Kato T et al (2005) Atrial natriuretic peptide promotes cardiomyocyte survival by cGMP-dependent nuclear accumulation of zyxin and Akt. J Clin Invest 115:2716-2730.

Zinchuk V et al (2005) Experimental LPS-induced cholestasis alters subcellular distribution and affects colocalization of Mrp2 and Bsep proteins: a quantitative colocalization study. Microsc Res Tech 67:65-70.

Zinchuk O et al (2005) Direct action of platelet activating factor (PAF) induces eosinophil accumulation and enhances expression of PAF receptors in conjunctivitis. Mol Vis 11:114-123.

Criscuoli ML et al (2005) Tumor metastasis but not tumor growth is dependent on Src-mediated vascular permeability. Blood 105:1508-1514.

2004

Zinchuk O et al (2004) Dynamics of PAF-induced conjunctivitis reveals differential expression of PAF-receptor by macrophages and eosinophils. Cell Tissue Res 317:265-277.

Related reading on the theory of quantitative colocalization:

Carlsson (1990) Scanning and detection techniques used in a confocal scanning laser microscope. J Microsc 157:21-27.

Mossberg K and Ericsson M (1990) Detection of doubly stained fluorescent specimens using confocal microscopy. J Microsc 158:215-224.

Mossberg K et al (1990) Computerized quantification of immunofluorescence labeled axon terminals and of co-localization of neurochemicals in axon terminals with a confocal scanning laser microscope. J Histochem Cytochem 38:179-190.

Manders EMM et al (1992) Dynamics of three-dimensional replication patterns during the S-phase, analysed by double labelling of DNA and confocal microscopy. J Cell Sci 103:857-862.

Manders EMM et al (1993) Measurement of co-localization of objects in dual-color confocal images. J Microsc 169:375-382.

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