Colocalization
Encyclopedia
In fluorescence microscopy, colocalization refers to observation of the spatial overlap between two (or more) different fluorescent labels, each having a separate emission wavelength, to see if the different "targets" are located in the same area of the cell or very near to one another. The definition can be split into two different phenomena, co-occurrence, which refers to the presence of two (possibly unrelated) fluorophores in the same pixel, and correlation, a much more significant statistical relationship between the fluorophores indicative of a biological interaction.. This technique is important to many cell biological and physiological studies during the demonstration of a relationship between pairs of bio-molecules.
to microscopists, along with other coefficients of which the "overlap coefficients" M1 and M2 have proved to be the most popular and useful Manders et al (1993). "Measurement of co-localisation of objects in dual-colour confocal images. Journal of Microscopy 169:375-382 . The purpose of using coefficients is to characterize the degree of overlap between images, usually two channels in a multidimensional microscopy image recorded at different emission wave lengths. A popular approach was introduced by Sylvain Costes, who utilized Pearson's Correlation Coefficient as a tool for setting the thresholds required by M1 and M2 in an objective fashion Costes et al (2004) "Automatic and Quantitative Measurement of Protein-Protein Colocalization in Live Cells." http://www.cell.com/biophysj/abstract/S0006-3495(04)74439-2. Costes approach makes the assumption that only positive correlations are of interest, and does not provide a useful measurement of PCC.
Although the use of coefficients can significantly improve the reliability of colocalization detection, it depends on the number of factors, including the conditions of how samples with fluorescence were prepared and how images with colocalization were acquired and processed. Studies should be conducted with great caution, and after careful background reading. Currently the field is dogged by confusion and a standardized approach is yet to be firmly established BOLTE and CORDELIÈRES (2006) "A guided tour into subcellular colocalization analysis in light microscopy." http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2818.2006.01706.x/abstract. Attempts to rectify this include re-examination and revision of some of the coefficients Adler and Parmryd (2010)"Quantifying colocalization by correlation: The Pearson correlation coefficient is superior to the Mander's overlap coefficient." http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.20896/abstract, application of a factor to correct for noise, and the proposal of further protocols which were thoroughly reviewed by Bolte and Cordelieres (2006)BOLTE and CORDELIÈRES (2006) "A guided tour into subcellular colocalization analysis in light microscopy." http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2818.2006.01706.x/abstract. In addition, due to the tendency of fluorescence images to contain a certain amount of out-of-focus signal, and poisson shot and other noise, they usually require pre-processing prior to quantification.. Careful image restoration by deconvolution removes noise and increases contrast in images, improving the quality of colocalization analysis results.
and nuclei
of apoptizing
and necrotizing cells among each of four different tissue types examined. Namely: the cerebral cortex
, the hippocampus
, the cerebellum
, and it was also demonstrated that colocalized detection of zinc increase and the well accepted cell death indicator propidium iodide also occurred in kidney cells. Using the principles of fluorescent colocalization. coincident detection of zinc accumulation and propidium iodide (a traditional cell death indicator) uptake in multiple cell types was demonstrated. (Stork & Li, The Journal of Neuroscience Methods, 2006). Various examples of quantification of colocalization in the field of neuroscience can be found in a review.
History of Colocalization
The ability to demonstrate a correlation between a pair of bio-molecules was greatly enhanced by Erik Manders of the University of Amsterdam who introduced Pearson's Correlation CoefficientPearson product-moment correlation coefficient
In statistics, the Pearson product-moment correlation coefficient is a measure of the correlation between two variables X and Y, giving a value between +1 and −1 inclusive...
to microscopists, along with other coefficients of which the "overlap coefficients" M1 and M2 have proved to be the most popular and useful Manders et al (1993). "Measurement of co-localisation of objects in dual-colour confocal images. Journal of Microscopy 169:375-382 . The purpose of using coefficients is to characterize the degree of overlap between images, usually two channels in a multidimensional microscopy image recorded at different emission wave lengths. A popular approach was introduced by Sylvain Costes, who utilized Pearson's Correlation Coefficient as a tool for setting the thresholds required by M1 and M2 in an objective fashion Costes et al (2004) "Automatic and Quantitative Measurement of Protein-Protein Colocalization in Live Cells." http://www.cell.com/biophysj/abstract/S0006-3495(04)74439-2. Costes approach makes the assumption that only positive correlations are of interest, and does not provide a useful measurement of PCC.
Although the use of coefficients can significantly improve the reliability of colocalization detection, it depends on the number of factors, including the conditions of how samples with fluorescence were prepared and how images with colocalization were acquired and processed. Studies should be conducted with great caution, and after careful background reading. Currently the field is dogged by confusion and a standardized approach is yet to be firmly established BOLTE and CORDELIÈRES (2006) "A guided tour into subcellular colocalization analysis in light microscopy." http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2818.2006.01706.x/abstract. Attempts to rectify this include re-examination and revision of some of the coefficients Adler and Parmryd (2010)"Quantifying colocalization by correlation: The Pearson correlation coefficient is superior to the Mander's overlap coefficient." http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.20896/abstract, application of a factor to correct for noise, and the proposal of further protocols which were thoroughly reviewed by Bolte and Cordelieres (2006)BOLTE and CORDELIÈRES (2006) "A guided tour into subcellular colocalization analysis in light microscopy." http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2818.2006.01706.x/abstract. In addition, due to the tendency of fluorescence images to contain a certain amount of out-of-focus signal, and poisson shot and other noise, they usually require pre-processing prior to quantification.. Careful image restoration by deconvolution removes noise and increases contrast in images, improving the quality of colocalization analysis results.
Examples of use
Some impermeable fluorescent zinc dyes can detectably label the cytosolCytosol
The cytosol or intracellular fluid is the liquid found inside cells, that is separated into compartments by membranes. For example, the mitochondrial matrix separates the mitochondrion into compartments....
and nuclei
Cell nucleus
In cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these...
of apoptizing
Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
and necrotizing cells among each of four different tissue types examined. Namely: the cerebral cortex
Cerebral cortex
The cerebral cortex is a sheet of neural tissue that is outermost to the cerebrum of the mammalian brain. It plays a key role in memory, attention, perceptual awareness, thought, language, and consciousness. It is constituted of up to six horizontal layers, each of which has a different...
, the hippocampus
Hippocampus
The hippocampus is a major component of the brains of humans and other vertebrates. It belongs to the limbic system and plays important roles in the consolidation of information from short-term memory to long-term memory and spatial navigation. Humans and other mammals have two hippocampi, one in...
, the cerebellum
Cerebellum
The cerebellum is a region of the brain that plays an important role in motor control. It may also be involved in some cognitive functions such as attention and language, and in regulating fear and pleasure responses, but its movement-related functions are the most solidly established...
, and it was also demonstrated that colocalized detection of zinc increase and the well accepted cell death indicator propidium iodide also occurred in kidney cells. Using the principles of fluorescent colocalization. coincident detection of zinc accumulation and propidium iodide (a traditional cell death indicator) uptake in multiple cell types was demonstrated. (Stork & Li, The Journal of Neuroscience Methods, 2006). Various examples of quantification of colocalization in the field of neuroscience can be found in a review.
Related techniques
- Förster resonance energy transfer (FRET): 10 nm proximity
- (Light microscopy: only 250 nm resolution; no certainty of effective interaction)
- Immuno precipitation (IP) dropdowns / pulldowns
- Yeast 2 hybrid - protein interaction mapping
open source
- FIJI is just ImageJ - batteries included http://pacific.mpi-cbg.de/wiki/index.php/Colocalization_Analysis
- BioImage XD http://www.bioimagexd.net/
closed source
- AxioVision Colocalization Module http://www.zeiss.de/C12567BE0045ACF1/ContentsWWWIntern/BBBE461B246BC593C12571AE003BEC98
- Colocalization Research Software http://www.colocalizer.com
- CoLocalizer Pro CoLocalizer ProCoLocalizer ProCoLocalizer Pro is a scientific software application, developed by , that allows researchers to analyze colocalization in the images obtained using fluorescence microscopy. Due to high popularity of Macintosh computers in medicine and biology, the software is designed specifically for Mac OS X...
- Elements Colocalization Module http://www.nis-elements.com/compare.html
- Huygens Colocalization Analyzer http://www.svi.nl/support/wiki/ColocalizationAnalyzer
- Volocity http://www.cellularimaging.com/products/Volocity/
- Volocity Demo http://www.cellularimaging.com/products/volocity/demo/
- Image Pro http://www.mediacy.com/index.aspx?page=IPP
- Bitplane Imaris http://www.bitplane.com