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

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Confocal Microscopy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1075))

Abstract

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.

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Author information

Authors and Affiliations

  1. Advanced Light Microscopy Unit, Centre for Genomic Regulation, Barcelona, Spain

    Timo Zimmermann

  2. Imaging and Cytometry Laboratory, Technology Facility, Department of Biology, University of York, York, UK

    Joanne Marrison, Karen Hogg & Peter O’Toole

Authors
  1. Timo Zimmermann
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  2. Joanne Marrison
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  3. Karen Hogg
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  4. Peter O’Toole
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Editor information

Editors and Affiliations

  1. University of Wisconsin Dept. of Molecular Biology, Madison, Wisconsin, USA

    Stephen W. Paddock

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Zimmermann, T., Marrison, J., Hogg, K., O’Toole, P. (2014). Clearing Up the Signal: Spectral Imaging and Linear Unmixing in Fluorescence Microscopy. In: Paddock, S. (eds) Confocal Microscopy. Methods in Molecular Biology, vol 1075. Humana Press, New York, NY. https://doi.org/10.1007/978-1-60761-847-8_5

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  • DOI: https://doi.org/10.1007/978-1-60761-847-8_5

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-58829-351-0

  • Online ISBN: 978-1-60761-847-8

  • eBook Packages: Springer Protocols

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