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Colocalization is defined as the presence of two or more color types of fluorescently-labeled 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 and 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.

Coefficients calculated by CoLocalizer and their significance

Pearson's correlation coefficient Rr 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:

Rr Coefficient Algorithm

Manders’ overlap coefficient R indicates an overlap of the signals. This coefficient is not sensitive to the limitations of typical fluorescence imaging, such as efficiency of hybridization, sample photobleaching, and camera quantum efficiency:

R Coefficient Algorithm

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:

K1 Coefficient Algorithm
K2 Coefficient Algorithm

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:

m1 Coefficient Algorithm
m1 Coefficient Algorithm

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:

M1 Coefficient Algorithm
M2 Coefficient Algorithm

Studies performed with the help of CoLocalizer software 

 

2017 

Thorpe RB et al (2017) Chronic hypoxia attenuates the vasodilator efficacy of protein kinase G in fetal and adult ovine cerebral arteries. Am J Physiol Heart Circ Physiol 313:H207-H219.

Silpanisong J et al (2017) Chronic hypoxia alters fetal cerebrovascular responses to endothelin1. Am J Physiol Cell Physiol 313:C207-C218.

 

2016 

Pearce WJ et al (2016) Imatinib attenuates cerebrovascular injury and phenotypic transformation after intracerebral hemorrhage in rats. Am J Physiol Regul Integr Comp Physiol 311: R1093–R1104.

Sjölander J et al (2016) C4b-binding protein protects β-cells from islet amyloid polypeptide induced cytotoxicity. J Biol Chem 291:21644-21655.

Wada T et al (2016) Nuclear cereblon modulates transcriptional activity of Ikaros and regulates its downstream target, enkephalin, in human neuroblastoma cells. Biochem Biophys Res Commun 477:388-394.

Venkatesh A et al (2016) TSC but not PTEN loss in starving cones of retinitis pigmentosa mice leads to an autophagy defect and mTORC1 dissociation from the lysosome. Cell Death & Disease 7 e2279; doi:10.1038/cddis.2016.182.

Martinez NA et al (2016) Caveolin-1 regulates the P2Y2 receptor signaling in human 1321N1 astrocytoma cells. J Biol Chem 291:12208-12222.

Li L-F et al (2016) Guanylate-binding protein 1, an interferon-induced GTPase, exerts an antiviral activity against classical swine fever virus depending on its GTPase activity. J Virol 90:4412-4426.

Zieger M and Punzo C (2016) Improved cell metabolism prolongs photoreceptor survival upon retinal-pigmented epithelium loss in the sodium iodate induced model of geographic atrophy. Oncotarget 9:9620-9633.

Figueiredo MA et al (2016) High level of GHR nuclear translocation in skeletal muscle of a hyperplasic transgenic zebrafish. J Mol Endocrinol 56:47-54.

 

2015 

Huang L et al (2015) A sequence of basic residues in the porcine circovirus type 2 capsid protein is crucial for its co-expression and co-localization with the replication protein. J Gen Virol 96:3566-3576.

Wang J et al (2015) Paclitaxel tumor priming promotes delivery and transfection of intravenous lipid-siRNA in pancreatic tumors. J Control Release 216:103-110.

Fénéant L et al (2015) New insights into the understanding of hepatitis C virus entry and cell-to-cell transmission by using the ionophore Monensin A. J Virol 89:8346-8364.

Li S et al (2016) Thioredoxin 2 is a novel E2-interacting protein that inhibits the replication of classical swine fever virus. J Virol 89:8510-8524.

Rassam P et al (2015) Supramolecular assemblies underpin turnover of outer membrane proteins in bacteria. Nature 523:333-336.

Venkatesh A et al (2015) Activated mTORC1 promotes long-term cone survival in retinitis pigmentosa mice. J Clin Invest 125:1446–1458.

 

2014 

Wang Q et al (2014) A recombinant avian leukosis virus subgroup J for directly monitoring viral infection and the selection of neutralizing antibodies. PLOS ONE 10:e0115422.

Liu F et al (2014) Metabotropic gludtamate receptor 5 modulates calcium oscillation and innate immune response induced by lipopolysaccharide in microglial cell. Neuroscience 281:24–34.

Kägebein D et al (2014) Chlamydia-infected epithelial cells and fibroblasts retain the ability to express surface-presented MHC class I molecules. Infect Immun 82:993-1006.

Zinchuk V and Grossenbacher-Zinchuk O (2014) Quantitative colocalization analysis of fluorescence microscopy images. Curr Protoc Cell Biol 62:4.19.14.19.14. (PROTOCOL)

Kazi JU et al (2014) Src-Like adaptor protein (SLAP) differentially regulates normal and oncogenic c-Kit signaling. J Cell Sci 127:653-662.

He F et al (2014) Beta-actin interacts with the E2 protein and is involved in the early replication of classical swine fever virus. Virus Res 179:161–168.

Güttsches A-K et al (2014) Human -defensin-3 correlates with muscle fibre degeneration in idiopathic inflammatory myopathies. Innate Immun 20:49-60.

 

2013 

Otteby KE et al (2013) Cartilage oligomeric matrix protein-induced complement activation in systemic sclerosis. Arthritis Res Ther 15:R215.

Tseng L T-L et al (2013) LMBD1 protein serves as a specific adaptor for insulin receptor internalization. J Biol Chem 288:32424-32432.

Head BP et al (2013) Neuronal specific targeting of cave-in expression to restore synaptic signalling and improve cognitive function in the neurodegenerative brain and motor function in spinal cord. United States Patent Application 20130224163.

Burchell VS et al (2013) The Parkinson’s disease genes Fbxo7 and Parkin interact to mediate mitophagy. Nat Neurosci 16:1257-1265.

Potel J et al (2013) EWI-2wint promotes CD81 clustering that abrogates Hepatitis C Virus entry. Cell Microbiol 7:1234-1252.

Cummins TD et al (2013) Utilization of fluorescent probes for the quantification and identification of subcellular proteomes and biological processes regulated by lipid peroxidation products. Free Radic Biol Med 59:56–68.

Sarin M et al (2013) Alterations in c-Myc phenotypes resulting from dynamin-related protein 1 (Drp1)-mediated mitochondrial fission. Cell Death Dis 4:e670.

Kazi JU & Rönnstrand L (2013) Suppressor of cytokine signaling 2 (SOCS2) associates with FLT3 and negatively regulates downstream signaling. Mol Oncol 7:693-703.

Bath C (2013) Human corneal epithelial subpopulations: oxygen dependent ex vivo expansion and transcriptional profiling. Acta Ophthalmol 91:1-34.

Li D et al (2013) Hemoglobin subunit beta interacts with the capsid protein and antagonizes the growth of classical swine fever virus. J Virol 87:5707-5717.

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

Fiegl D 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) -catenin-dependent lysosomal targeting of internalized tumor necrosis factor- 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-B 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 L 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 ABA et al (2011) SMS1-generated sphingomyelin plays an important role in transferrin trafficking and cell proliferation. J Biol Chem 286:36053-36062.

Lok C-N 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 1 integrin enhances TGF-b1-induced collagen expression in epithelial cells via increased V3-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 2 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 -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.

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 for 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 TGF1-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 -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 -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.

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