Spectral unmixing methods and tools for the detection and quantitation of collagen and other macromolecules in tissue specimens

Zachary T. Harmany, Farzad Fereidouni, Richard M Levenson

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Collagen and other components in the extracellular matrix are proving of increasing importance for the understanding of complex cell and tissue interactions in a variety of settings. Detection and quantitation of these components can still prove challenging, and a number of techniques have been developed. We focus here on methods in fluorescence-based assessments, including multiplexed immunodetection and the use of simpler histochemical stains, both complemented by linear unmixing techniques. Typically, differentiating these components requires the use of a set of optical filters to isolate each fluorescent compound from each other and from often bright background autofluorescence signals. However, standard fluorescent microscopes are usually only able to separate a limited number of components. If the emission spectra of the fluorophores are spectrally distinct, but overlapping, sophisticated spectral imaging or computational methods can be used to optimize separation and quantitation. This chapter describes spectral unmixing methodology and associated open-source software tools available to analyze multispectral as well as simple color (RGB) images.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages491-509
Number of pages19
Volume1627
DOIs
StatePublished - 2017

Publication series

NameMethods in Molecular Biology
Volume1627
ISSN (Print)1064-3745

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Keywords

  • Fluorescent imaging
  • Image analysis
  • Linear unmixing
  • Microscopy
  • Multispectral imaging
  • Spectroscopy

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Harmany, Z. T., Fereidouni, F., & Levenson, R. M. (2017). Spectral unmixing methods and tools for the detection and quantitation of collagen and other macromolecules in tissue specimens. In Methods in Molecular Biology (Vol. 1627, pp. 491-509). (Methods in Molecular Biology; Vol. 1627). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-7113-8_30