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Optics Express

Optics Express

  • Editor: Andrew M. Weiner
  • Vol. 22, Iss. 3 — Feb. 10, 2014
  • pp: 2973–2988

Fluorescence spectral correlation spectroscopy (FSCS) for probes with highly overlapping emission spectra

Aleš Benda, Peter Kapusta, Martin Hof, and Katharina Gaus  »View Author Affiliations


Optics Express, Vol. 22, Issue 3, pp. 2973-2988 (2014)
http://dx.doi.org/10.1364/OE.22.002973


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Abstract

We present a fluorescence correlation spectroscopy (FCS) approach to obtain spectral cross-talk free auto- and cross-correlation functions for probes with highly overlapping emission spectra. Confocal microscopes with either a hyperspectral EM-CCD or six-channel PMT array spectral detection were used, followed by a photon filtering correlation approach that results in spectral unmixing. The method is highly sensitive and can distinguish between Atto488 and Oregon Green 488 signals so that auto-correlation curves can be fitted without the need for cross-talk correction. We also applied the approach to the membrane dye Laurdan whose emission is dependent on the lipid order within the bilayer. With fluorescence spectral correlation spectroscopy (FSCS), we could obtain spectral cross-talk free auto- and cross-correlation functions corresponding to Laurdan located in liquid ordered and liquid disordered phases.

© 2014 Optical Society of America

OCIS Codes
(170.6280) Medical optics and biotechnology : Spectroscopy, fluorescence and luminescence
(180.1790) Microscopy : Confocal microscopy
(300.2530) Spectroscopy : Fluorescence, laser-induced
(110.4234) Imaging systems : Multispectral and hyperspectral imaging

ToC Category:
Spectroscopy

History
Original Manuscript: January 20, 2014
Manuscript Accepted: January 20, 2014
Published: January 31, 2014

Virtual Issues
Vol. 9, Iss. 4 Virtual Journal for Biomedical Optics

Citation
Aleš Benda, Peter Kapusta, Martin Hof, and Katharina Gaus, "Fluorescence spectral correlation spectroscopy (FSCS) for probes with highly overlapping emission spectra," Opt. Express 22, 2973-2988 (2014)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-22-3-2973


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