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added notes from the AOMF seminar on colocalization
from http://www.aomf.ca/OMUGsummary.html#2June2010
June 2, 2008 - ImageJ/Colocalization - top
Introduction:
One of the most useful aspects of immunocytochemistry is the ability to compare the location of two or more proteins tagged with different fluorophores between or within cells. The routine method for doing this is to stain (or pseudostain) one fluorescent image red, the other green and look for yellow in the superimposed image. While this is an invaluable screening tool there are major limitations that are often not considered. Further, the method is often applied without thought as to whether it will answer the critical question. Thus, if one is simply interested in the question 'are the two proteins are located in the same region of the cell or the same cell' this approach may be sufficient. However, if the question is 'are these two proteins parts of a common complex' this method can be woefully insufficient and even totally misleading. These concepts will be introduced and debated and some methods that have been devised that may improve on the simple dye-overlay will be discussed.
References:
1. Bolte, S. and Cordelieres, F. P. A guided tour of subcellular colocalization analysis in lilght microscopy. Journal of Microscopy, Vol. 224, Pt3 December 2006, pp.213-232. (pdf)
2. Li, Q., Lau, A., Morris, T. J., Guo, L., Fordyce, C. B., and Stanley, E. F. (2004). A Syntaxin 1, Galphao, and N-Type Calcium Channel Complex at a Presynaptic Nerve Terminal: Analysis by Quantitative Immunocolocalization. J. Neurosci. 24, 4070-4081. (pdf)
June 2, 2008 - ImageJ/Colocalization - top
Introduction:
One of the most useful aspects of immunocytochemistry is the ability to compare the location of two or more proteins tagged with different fluorophores between or within cells. The routine method for doing this is to stain (or pseudostain) one fluorescent image red, the other green and look for yellow in the superimposed image. While this is an invaluable screening tool there are major limitations that are often not considered. Further, the method is often applied without thought as to whether it will answer the critical question. Thus, if one is simply interested in the question 'are the two proteins are located in the same region of the cell or the same cell' this approach may be sufficient. However, if the question is 'are these two proteins parts of a common complex' this method can be woefully insufficient and even totally misleading. These concepts will be introduced and debated and some methods that have been devised that may improve on the simple dye-overlay will be discussed.
References:
1. Bolte, S. and Cordelieres, F. P. A guided tour of subcellular colocalization analysis in lilght microscopy. Journal of Microscopy, Vol. 224, Pt3 December 2006, pp.213-232. (pdf)
2. Li, Q., Lau, A., Morris, T. J., Guo, L., Fordyce, C. B., and Stanley, E. F. (2004). A Syntaxin 1, Galphao, and N-Type Calcium Channel Complex at a Presynaptic Nerve Terminal: Analysis by Quantitative Immunocolocalization. J. Neurosci. 24, 4070-4081. (pdf)