Clearing up the signal: spectral imaging and linear unmixing in fluorescence microscopy

Timo Zimmermann; Joanne Marrison; Karen Hogg; Peter O'Toole

doi:10.1007/978-1-60761-847-8_5

Clearing up the signal: spectral imaging and linear unmixing in fluorescence microscopy

Timo Zimmermann, Joanne Marrison, Karen Hogg, Peter O'Toole

Biology

Research output: Contribution to journal › Article › peer-review

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.

Original language	English
Pages (from-to)	129-148
Number of pages	20
Journal	Methods in Molecular Biology
Volume	1075
DOIs	https://doi.org/10.1007/978-1-60761-847-8_5
Publication status	Published - 24 Aug 2014

Keywords

Fluorescence
Humans
Image Processing, Computer-Assisted
Microscopy, Fluorescence

Access to Document

10.1007/978-1-60761-847-8_5

Cite this

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title = "Clearing up the signal: spectral imaging and linear unmixing in fluorescence microscopy",

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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KW - Image Processing, Computer-Assisted

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