Multispectral analysis tools can increase utility of RGB color images in histology

Farzad Fereidouni, Croix Griffin, Austin Todd, Richard M Levenson

Research output: Contribution to journalArticlepeer-review

11 Scopus citations


Multispectral imaging (MSI) is increasingly finding application in the study and characterization of biological specimens. However, the methods typically used come with challenges on both the acquisition and the analysis front. MSI can be slow and photon-inefficient, leading to long imaging times and possible phototoxicity and photobleaching. The resulting datasets can be large and complex, prompting the development of a number of mathematical approaches for segmentation and signal unmixing. We show that under certain circumstances, just three spectral channels provided by standard color cameras, coupled with multispectral analysis tools, including a more recent spectral phasor approach, can efficiently provide useful insights. These findings are supported with a mathematical model relating spectral bandwidth and spectral channel number to achievable spectral accuracy. The utility of 3-band RGB and MSI analysis tools are demonstrated on images acquired using brightfield and fluorescence techniques, as well as a novel microscopy approach employing UV-surface excitation. Supervised linear unmixing, automated non-negative matrix factorization and phasor analysis tools all provide useful results, with phasors generating particularly helpful spectral display plots for sample exploration.

Original languageEnglish (US)
Article number044007
JournalJournal of Optics (United Kingdom)
Issue number4
StatePublished - Apr 1 2018


  • fluorescence
  • histopathology
  • phasor analysis
  • spectral imaging

ASJC Scopus subject areas

  • Electronic, Optical and Magnetic Materials
  • Atomic and Molecular Physics, and Optics


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