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 the 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 if so to which degree?", quantitative colocalization provides the answer. The degree of colocalization in medico-biological specimens can not be judged by the 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 quantification. 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 fluorescence microscopy images. These values are called colocalization coefficients.

See table for interpretation of the results of colocalization coefficients calculations.

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:

2013

Li D et al (2013) Hemoglobin subunit beta interacts with the capsid protein and antagonizes the growth of classical swine fever virus. J Virol in press.

Potel J et al (2013) EWI-2wint promotes CD81 clustering that abrogates Hepatitis C Virus entry. Cell Microbiol in press.

Bath C et al (2013) Hypoxia is a key regulator of limbal epithelial stem cell growth and differentiation. Stem Cell Res 10:349-360.

Ooms et al (2013) Development of a recombinant antibody to target peptides and proteins to sialoadhesin-expressing macrophages. BMC Biotechnol 13:33-43.

Fiegl et al (2013) Amphisomal route of MHC class I cross-presentation in bacteria-infected dendritic cells. J Immunol 190:2791-2806.

Zinchuk V et al (2013) Bridging the gap between qualitative and quantitative colocalization results in fluorescence microscopy studies. Sci Rep 3:1365 doi:10.1038/srep01365.

Han J et al (2013) β-catenin-dependent lysosomal targeting of internalized tumor necrosis factor-α suppresses caspase-8 activation in apoptosis-resistant colon cancer cells. Mol Biol Cell 24:465-473.

Lee MR et al (2013) Striatal adenosine signaling regulates EAAT2 and astrocytic AQP4 expression and alcohol drinking in mice. Neuropsychopharmacol 38:437-445.

2012

Kazi JU & Rönnstrand L (2012) Src-Like adaptor protein (SLAP) binds to the receptor tyrosine kinase Flt3 and modulates receptor stability and downstream signaling. PLOS ONE 7:e53509.

König H-G et al (2012) Fibroblast growth factor homologous factor 1 interacts with NEMO to regulate NF-κB signaling in neurons. J Cell Sci 125:6058-6070.

Martin M et al (2012) Annexin A2 and A5 serve as new ligands for C1q on apoptotic cells. J Biol Chem 287:33733-33744.

Teo L et al (2012) Discrete ephrin-B1 expression by specific layers of the primate retinogeniculostriate system continues throughout postnatal and adult life. J Comp Neurol 520:2941-2956.

Rohde J et al (2012) Orf virus interferes with MHC class I surface expression by targeting vesicular transport and Golgi. BMC Vet Res 8:114.

Graves JA et al (2012) Mitochondrial structure, function and dynamics are temporally controlled by c-Myc. PLOS ONE 7:e37699.

Bier C et al (2012) Overexpression of the catalytically impaired taspase1T234V or taspase1D233A variants does not have a dominant negative effect in T(4;11). PLOS ONE 7:e34142.

Sjölander J et al (2012) Islet amyloid polypeptide triggers limited complement activation and binds complement inhibitor C4b-binding protein, which enhances fibril formation. J Biol Chem 287:10824-10833.

Shen Z et al (2012) An artery-specific fluorescent dye for studying neurovascular coupling. Nat Meth 9:273-276.

Wu Y et al (2012) Critical evaluation of quantitative colocalization analysis in confocal fluorescence microscopy. Interdiscip Sci Comput Life Sci 4:27-37.

Pearn ML et al (2012) Propofol neurotoxicity is mediated by p75 neurotrophin receptor activation. Anesthesiology 116:352-361.

Canterini S et al (2012) Subcellular TSC22D4 localization in cerebellum granule neurons of the mouse depends on development and differentiation. Cerebellum 11:28-40.

2011

Belkacemi et al (2011) Altered mitochondrial apoptotic pathway in placentas from undernourished rat gestations. Am J Physiol Regul Integr Comp Physiol 301:R1599-R1615.

Yang L et al (2011) ncRNA- and Pc2 methylation-dependent gene relocation between nuclear structures mediates gene activation programs. Cell 147:773-788.

Zinchuk V et al (2011) Quantifying spatial correlations of fluorescent markers using enhanced background reduction with protein proximity index and correlation coefficient estimations. Nat Protoc 6:1554-1567.

Shakor et al (2011) SMS1-generated sphingomyelin plays an important role in transferrin trafficking and cell proliferation. J Biol Chem 286:36053-36062.

Lok C-M et al (2011) Activation of autophagy of aggregation-prone ubiquitinated proteins by timosaponin A-III. J Biol Chem 286:31684-31696.

Ganesean S et al (2011) Drosophila glutamate receptor mRNA expression and mRNP particles. RNA Biol 8:771-781.

Baumann C et al (2011) Chromatin configuration and epigenetic landscape at the sex chromosome bivalent during equine spermatogenesis. Chromosoma 120:227-244.

Remo BF et al (2011) Phosphatase-resistant gap junctions inhibit pathological remodeling and prevent arrhythmias. Circ Res 108:1459-1466.

Palatinus JA et al (2011) ZO-1 determines adherens and gap junction localization at intercalated disks. Am J Physiol Heart Circ Physiol 300:H583-H594.

Delputte PL et al (2011) Porcine sialoadhesin (CD169/Siglec-1) is an endocytic receptor that allows targeted delivery of toxins and antigens to macrophages. PLoS One 6:e16827.

Kuipers MA et al (2011) Highly stable loading of Mcm proteins onto chromatin in living cells requires replication to unload. J Cell Biol 192:29-41.

Chinzei R et al (2011) Vitamin K3 attenuates cerulein-induced acute pancreatitis through inhibition of the autophagic pathway. Pancreas 40:84-94.

Lemkuil BP et al (2011) Isoflurane neurotoxicity is mediated by p75TNR-RhoA activation and actin depolymerization. Anesthesiology 114:49-57.

2010

Krementsov D et al (2010) HIV-1 assembly differentially alters dynamics and partitioning of tetraspanins and Raft components. Traffic 11:1401-1414.

Hayashida T et al (2010) Loss of β1 integrin enhances TGF-b1-induced collagen expression in epithelial cells via increased αVβ3-integrin and RAC1 activity. J Biol Chem 285:30741-30751.

Delputte P et al (2010) Permissive cells and uses thereof. United States Patent Application 20100158947.

Tsutsumi Y et al (2010) Role of caveolin-3 and glucose transporter-4 in isoflurane-induced delayed cardiac protection. Anesthesiology 112:1136-1145.

Boileau AJ et al (2010) The short splice variant of the γ2 subunit acts as an external modulator of GABAA receptor function. J Neurosci 30:4895-4903.

Leffler J et al (2010) Annexin-II, DNA, and histones serve as factor H ligands on the surface of apoptotic cells. J Biol Chem 285:3766-3776.

2009

Zinchuk V and Grossenbacher-Zinchuk O (2009) Recent advances in quantitative colocalization analysis: focus on neuroscience. Prog Histochem Cytochem 44:125-172. (REVIEW).

Yousif L et al (2009) Mitochondria-penetrating peptides: sequence effects and model cargo transport. ChemBioChem 10:2081-2088.

Rubio M et al (2009) Cardioprotective stimuli mediate phosphoinositide 3-kinase and phosphoinositide dependent kinase 1 nuclear accumulation in cardiomyocytes. J Mol Cell Cardiol 47:96-103.

Katzenellenbogen RA et al (2009) NFX1-123 increases hTERT expression and telomerase activity posttranscriptionally in human papillomavirus type 16 E6 keratinocytes. J Virol 83:6446-6456.

Yoshizawa T et al (2009) Role of MAPK kinase 6 in arthritis: distinct mechanism of action in inflammation and cytokine expression. J Immunol 183:1360-1367.

Davies BL et al (2009) Lung respiratory rhythm and pattern generation in the bullfrog: role of neurokinin-1 and μ-opioid receptors. J Comp Physiol B 179:579-592.

Lehmann M et al (2009) Intracellular transport of human immunodeficiency virus type 1 genomic RNA and viral production are dependent on dynein motor function and late endosome positioning. J Biol Chem 284:14572-14585.

Horton K and Kelley S (2009) Engineered apoptosis-inducing peptides with enhanced mitochondrial localization and potency. J Med Chem 52:3293-3299.

Baumann C and De La Fuente R (2009). ATRX marks the inactive X chromosome (Xi) in somatic cells and during imprinted X chromosome inactivation in trophoblast stem cells. Chromosoma 118:209-222.

Head BP et al (2009) Inhibition of p75 neurotrophin receptor attenuates isoflurane-mediated neuronal apoptosis in the neonatal central nervous system. Anesthesiology 100:813-825.

Fujii R et al (2009) TLS-GFP cannot rescue mRNP formation near spines and spine phenotype in TLS-KO. Neuroreport 20:57-61.

2008

Yalcin S et al (2008) Foxo3 is essential for the regulation of ataxia telengiectasia mutated and oxidative stress-mediated homeostasis of hematopoietic stem cells. J Biol Chem 283:25692-25705.

Zinchuk V and Grossenbacher-Zinchuk O (2008) Quantitative colocalization analysis of confocal fluorescence microscopy images. Curr Protoc Cell Biol Chapter 4 Unit 4.19. (PROTOCOL)

Polyak MJ et al (2008) CD20 homo-oligomers physically associate with the B cell antigen receptor. J Biol Chem 283:18545-18552.

Ajamian L et al (2008) Unexpected roles for UPF1 in HIV-1 RNA metabolism and translation. RNA 14:914-927.

Horton KL et al (2008) Mitochondria-penetrating peptides. Chem Biol 15:375-382.

Head BP et al (2008) Caveolin-1 expression is essential fo N-methyl-D-aspartate receptor-mediated Src and extracellular signal-regulated kinase 1/2 activation and protection of primary neurons from ischemic death. FASEB J 22:828-840.

2007

Ma et al (2007) DNA-passivated CdS nanocrystals: luminescence, bioimaging, and toxicity profiles. Langmuir 23:12783-12787.

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

Hayashida T et al (2007) MAP-kinase activity necessary for TGFβ1-stimulated mesangial cell type I collagen expression resuired adhesion-dependent phosphorylation of FAK tyrosine 397. J Cell Sci 120:4230-4240.

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. (REVIEW)

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

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. (REVIEW)

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.

Smallcombe A (2001) Multicolor imaging: the important question of co-localization. Biotechniques 30:1240–1246.

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