Clearing Up the Signal: Spectral Imaging and Linear Unmixing in Fluorescence Microscopy

  • Timo Zimmermann
  • Joanne Marrison
  • Karen Hogg
  • Peter O’Toole
Part of the Methods in Molecular Biology book series (MIMB, volume 1075)


The ongoing progress in fluorescence labeling and in microscope instrumentation allows the generation and the imaging of complex biological samples that contain increasing numbers of fluorophores. For the correct quantitative analysis of datasets with multiple fluorescence channels, it is essential that the signals of the different fluorophores are reliably separated. Due to the width of fluorescence spectra, this cannot always be achieved using the fluorescence filters in the microscope. In such cases spectral imaging of the fluorescence data and subsequent linear unmixing allows the separation even of highly overlapping fluorophores into pure signals. In this chapter, the problems of fluorescence cross talk are defined, the concept of spectral imaging and separation by linear unmixing is described, and an overview of the microscope types suitable for spectral imaging are given.

Key words

Spectral imaging Linear unmixing Image analysis Fluorescence cross talk Multichannel imaging 


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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Timo Zimmermann
    • 1
  • Joanne Marrison
    • 2
  • Karen Hogg
    • 2
  • Peter O’Toole
    • 2
  1. 1.Advanced Light Microscopy UnitCentre for Genomic RegulationBarcelonaSpain
  2. 2.Imaging and Cytometry Laboratory, Technology Facility, Department of BiologyUniversity of YorkYorkUK

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