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Optics Express

Optics Express

  • Editor: Michael Duncan
  • Vol. 13, Iss. 19 — Sep. 19, 2005
  • pp: 7415–7423

Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) with low background and high count-rate per molecule

Kai Hassler, Marcel Leutenegger, Per Rigler, Ramachandra Rao, Rudolf Rigler, Michael Gösch, and Theo Lasser  »View Author Affiliations


Optics Express, Vol. 13, Issue 19, pp. 7415-7423 (2005)
http://dx.doi.org/10.1364/OPEX.13.007415


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Abstract

We designed a fluorescence correlation spectroscopy (FCS) system for measurements on surfaces. The system consists of an objective-type total internal reflection fluorescence (TIRF) microscopy setup, adapted to measure FCS. Here, the fluorescence exciting evanescent wave is generated by epi-illumination through the periphery of a high NA oil-immersion objective. The main advantages with respect to conventional FCS systems are an improvement in terms of counts per molecule (cpm) and a high signal to background ratio. This is demonstrated by investigating diffusion as well as binding and release of single molecules on a glass surface. Furthermore, the size and shape of the molecule detection efficiency (MDE) function was calculated, using a wave-vectorial approach and taking into account the influence of the dielectric interface on the emission properties of fluorophores.

© 2005 Optical Society of America

OCIS Codes
(170.6280) Medical optics and biotechnology : Spectroscopy, fluorescence and luminescence
(240.6490) Optics at surfaces : Spectroscopy, surface
(300.2530) Spectroscopy : Fluorescence, laser-induced

ToC Category:
Research Papers

History
Original Manuscript: July 22, 2005
Revised Manuscript: September 2, 2005
Published: September 19, 2005

Citation
Kai Hassler, Marcel Leutenegger, Per Rigler, Ramachandra Rao, Rudolf Rigler, Michael Gösch, and Theo Lasser, "Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) with low background and high count-rate per molecule," Opt. Express 13, 7415-7423 (2005)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-13-19-7415


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