Co-localization Short description of the method Co-localization is defined by the presence of two or more different molecules at the same location in a specimen. However, in the context of digital imaging, the term co-localization refers to colors emitted by fluorescent molecules detected in the same pixel in the image. It is important to be aware of the fact that co-localization can not be analyzed for fluorophores with similar emission spectra. Accurate co-localization analysis is only possible if the fluorescence emission spectra are well separated between fluorophores and the correct filter sets are selected or by using linear unmixing for data acquisition. Qualitative analysis can be thought of as “highlighting overlapping pixels”. Although this is often given as a number (“percentage overlap”) suggesting quantification, the qualitative aspect arises when the user has to define what is considered “overlap- ping”. The two channels have a threshold set and any areas of overlap are considered “co-localised”. Quantitative analysis compensate subjective interpretation of users by analysing all the pixels based on their intensity. This result is shown in a scatter plot, a type of mathematical diagram to display values for two variables (fluorescence intensities of two channels) for a set of data. Out of this plot a number of coefficients can be determined, which describe the degree of co-localization. Possible applications In fluorescence microscopy, co-localization is a useful tool for determining the levels of two fluorescent probes binding at the same apparent physical location within a cell or sub-cellular structure. Obtaining relevant qualitative information in the form of overlapping regions is of interest to an investigator, as is the quantitative aspect of that overlap. Examples of applications in our group Matthas S et al., J Exp Med. 199 1041-1052 (2004) Jundt F et al., Leukemia 22 1587-1594 (2008) Bit-Avragim N et al., Dev Dyn. 237 83-90 (2008) Hava D. et al., J Cell Sci. 122 687-695 (2009) Schwefel D et al., Prod Natl Acad Sci USA 107 20299-20304 (2010) Otten C. et al., PLoS One 7 e31041 (2012) Bogum J et al., JACS in press .