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Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 16, Iss. 19 — Sep. 15, 2008
  • pp: 14353–14368

The optics and performance of dual-focus fluorescence correlation spectroscopy

Thomas Dertinger, Anastasia Loman, Benjamin Ewers, Claus B. Müller, Benedikt Krämer, and Jörg Enderlein  »View Author Affiliations


Optics Express, Vol. 16, Issue 19, pp. 14353-14368 (2008)
http://dx.doi.org/10.1364/OE.16.014353


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Abstract

Fluorescence correlation spectroscopy (FCS) is an important spectroscopic technique which can be used for measuring the diffusion and thus size of fluorescing molecules at pico- to nanomolar concentrations. Recently, we introduced an extension of conventional FCS, which is called dual-focus FCS (2fFCS) and allows absolute diffusion measurements with high precision and repeatability. It was shown experimentally that the method is robust against most optical and sample artefacts which are troubling conventional FCS measurements, and is furthermore able to yield absolute values of diffusion coefficients without referencing against known standards. However, a thorough theoretical treatment of the performance of 2fFCS is still missing. The present paper aims at filling this gap. Here, we have systematically studied the performance of 2fFCS with respect to the most important optical and photophysical factors such as cover slide thickness, refractive index of the sample, laser beam geometry, and optical saturation. We show that 2fFCS has indeed a superior performance when compared with conventional FCS, being mostly insensitive to most potential aberrations when working under optimized conditions.

© 2008 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

ToC Category:
Spectroscopy

History
Original Manuscript: July 3, 2008
Revised Manuscript: August 12, 2008
Manuscript Accepted: August 13, 2008
Published: August 29, 2008

Virtual Issues
Vol. 3, Iss. 11 Virtual Journal for Biomedical Optics

Citation
Thomas Dertinger, Anastasia Loman, Benjamin Ewers, Claus B. Müller, Benedikt Krämer, and Jörg Enderlein, "The optics and performance of dual-focus fluorescence correlation spectroscopy," Opt. Express 16, 14353-14368 (2008)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-16-19-14353


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