Linear fluorescence unmixing in cell biological research

One of the most important issues in biomedical research microscopy is the analysis of the spatial localization of fluorescently labelled structures. Today, a wide range of available dyes, fluorescent proteins and labelling techniques allows the creation of complex multicolored samples to study intracellular localization. However, analysis of localization and colocalization is often perturbed by a significant overlap of the fluorophores used to label the structures. This problem can be addressed by the use of multichannel fluorescence imaging together with linear unmixing of the image data. This method allows the reliable separation of overlapping fluorescence signals and subsequent accurate and quantitative (co)-localization analysis. In this chapter, we explain the theory of linear unmixing, provide information on how to perform such experiments, and point out technical limitations and potential pitfalls. Finally we demonstrate the benefit of this methodology, employing examples from cell biology and virology.