Colocalization
In
History
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
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.[6] Attempts to rectify this include re-examination and revision of some of the coefficients,[7][8] application of a factor to correct for noise,[1] "Replicate based noise corrected correlations for accurate measurements of colocalization".[9] and the proposal of further protocols,[10] which were thoroughly reviewed by Bolte and Cordelieres (2006).[6] 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.[11][12] Careful image restoration by deconvolution removes noise and increases contrast in images, improving the quality of colocalization analysis results. Up to now, most frequently used methods to quantify colocalization calculate the statistical correlation of pixel intensities in two distinct microscopy channels. More recent studies have shown that this can lead to high correlation coefficients even for targets that are known to reside in different cellular compartments.[13] A more robust quantification of colocalization can be achieved by combining digital object recognition, the calculation of the area overlap and combination with a pixel-intensity correlation value. This led to the concept of an object-corrected Pearson's correlation coefficient.[13]
Examples of use
Some impermeable fluorescent zinc dyes can detectably label the
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 [17]
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.[18]
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
open source
- FIJI is just ImageJ - batteries included
- BioImage XD
closed source
- AxioVision Colocalization Module
- Colocalization Research Software
- CoLocalizer Pro CoLocalizer Pro
- Nikon's NIS-Elements Colocalization Module
- Scientific Volume Imaging's Huygens Colocalization Analyzer
- Quorum Technology's Volocity
- Media Cybernetics's Image-Pro
- Bitplane's Imaris
- arivis Vision4D
- [19]
References
- ^ a b Adler et al. (2008)
- ^ Manders et al (1992). "Dynamics of three-dimensional replication patterns during the S-phase, analysed by double labelling of DNA and confocal microscopy." [1]
- S2CID 95098323.
- ^ Zinchuk V et al (2007). "Quantitative colocalization analysis of multicolor confocal immunofluorescence microscopy images: pushing pixels to explore biological phenomena". Acta Histochem Cytochem 40:101-111.
- ^ Costes et al (2004) "Automatic and Quantitative Measurement of Protein-Protein Colocalization in Live Cells." [2]
- ^ a b BOLTE and CORDELIÈRES (2006) "A guided tour into subcellular colocalization analysis in light microscopy." [3]
- ^ Adler and Parmryd (2010)"Quantifying colocalization by correlation: The Pearson correlation coefficient is superior to the Mander's overlap coefficient." [4]
- ^ Krauß et al (2015). "Colocalization of fluorescence and Raman microscopic images for the identification of subcellular compartments: a validation study." Analyst, volume 140, issue 7, pages 2360-2368. [5]
- S2CID 12758752.
- ^ Curr Protoc Cell Biol "Quantitative colocalization analysis of confocal fluorescence microscopy images." Archived 2009-11-28 at the Wayback Machine
- ^ Pawley JB (2006). Handbook of Biological Confocal Microscopy
- ^ 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.
- ^ PMID 27420480.
- S2CID 16900662.
- ^ Zinchuk V & Grossenbacher-Zinchuk O (2009). "Recent advances in quantitative colocalization analysis: Focus on neuroscience". Prog Histochem Cytochem 44:125-172
- ^ "Adler J & Parmryd I (2013) Methods Mol Biol 931, 97-109". Colocalization analysis in fluorescence microscopy. Retrieved 2016-04-19.
- PMID 22341441.
- PMID 23455567.
- ^ Rewire Neuro's Pipsqueak Pro