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Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 20, Iss. 12 — Jun. 4, 2012
  • pp: 12729–12741

Spectral phasor analysis allows rapid and reliable unmixing of fluorescence microscopy spectral images

Farzad Fereidouni, Arjen N. Bader, and Hans C. Gerritsen  »View Author Affiliations

Optics Express, Vol. 20, Issue 12, pp. 12729-12741 (2012)

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A new global analysis algorithm to analyse (hyper-) spectral images is presented. It is based on the phasor representation that has been demonstrated to be very powerful for the analysis of lifetime imaging data. In spectral phasor analysis the fluorescence spectrum of each pixel in the image is Fourier transformed. Next, the real and imaginary components of the first harmonic of the transform are employed as X and Y coordinates in a scatter (spectral phasor) plot. Importantly, the spectral phasor representation allows for rapid (real time) semi-blind spectral unmixing of up to three components in the image. This is demonstrated on slides with fixed cells containing three fluorescent labels. In addition the method is used to analyse autofluorescence of cells in a fresh grass blade. It is shown that the spectral phasor approach is compatible with spectral imaging data recorded with a low number of spectral channels.

© 2012 OSA

OCIS Codes
(110.2960) Imaging systems : Image analysis
(170.2520) Medical optics and biotechnology : Fluorescence microscopy
(110.4234) Imaging systems : Multispectral and hyperspectral imaging

ToC Category:
Imaging Systems

Original Manuscript: March 5, 2012
Revised Manuscript: April 11, 2012
Manuscript Accepted: April 11, 2012
Published: May 22, 2012

Virtual Issues
Vol. 7, Iss. 8 Virtual Journal for Biomedical Optics

Farzad Fereidouni, Arjen N. Bader, and Hans C. Gerritsen, "Spectral phasor analysis allows rapid and reliable unmixing of fluorescence microscopy spectral images," Opt. Express 20, 12729-12741 (2012)

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