Colocalization

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.

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. ==Examples of use== Some impermeable fluorescent zinc dyes can detectably label the cytosol 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. Various examples of quantification of colocalization in the field of neuroscience can be found in a review. Detailed protocols on the quantification of colocalization can be found in a book chapter. ==Single-molecule resolution== Colocalization is used in real-time single-molecule fluorescence microscopy to detect interactions between fluorescently labeled molecular species. In this case, one species (e.g. a DNA molecule) is typically immobilized on the imaging surface, and the other species (e.g. a DNA-binding protein) is supplied to the solution. The two species are labeled with dyes of spectrally resolved (>50 nm) colors, e.g. cyanine-3 and cyanine-5. Fluorescence excitation is typically carried out in total internal reflection mode which increases the signal-to-noise ratio for the molecules at the surface with respect to the molecules in bulk solution. The molecules are detected as spots appearing on the surface in real-time, and their locations are found to within 10-20 nm by fitting of point-spread functions. Since typical sizes of biomolecules are on the order of 10 nm, this precision is usually sufficient for calling of molecular interactions ==Interpretation of results== For the purpose of better interpretation of the results of qualitative and quantitative colocalization studies, it was suggested to use a set of five linguistic variables tied to the values of colocalization coefficients, such as very weak, weak, moderate, strong, and very strong, for describing them. The approach is based on the use of the fuzzy system model and computer simulation. When new coefficients are introduced, their values can be fitted into the set. ==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 ==Benchmark images== The degree of colocalization in fluorescence microscopy images can be validated using the Colocalization Benchmark Source, a free collection of downloadable image sets with pre-defined values of colocalization. ==Software implementations==

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