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Biophys. J. BioFAST: First Published September 7, 2007. doi:10.1529/biophysj.107.115998
© 2007 by the Biophysical Society.

A more recent version of this article appeared on January 1, 2008.
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SPECTROSCOPY, IMAGING, OTHER TECHNIQUES

Supercritcal Angle Fluorescence Correlation Spectroscopy

Jonas Ries 1, Thomas Ruckstuhl 2, Dorinel Verdes 2 and Petra Schwille 1*

1 Biotec, TU Dresden
2 Institute of Physical Chemistry, University of Zurich

* To whom correspondence should be addressed. E-mail: schwille{at}biotec.tu-dresden.de.

Submitted on June 23, 2007
Revised on July 30, 2007
Accepted on 21 August 2007


  Abstract
We explore the potential of a supercritical angle objective for fluorescence correlation spectroscopy (FCS). This novel microscope objective combines tight focusing by an aspheric lens with strong axial confinement of supercritical angle fluorescence (SAF) collection by a parabolic mirror lens, resulting in a small detection volume. The tiny axial extent of the detection volume features an excellent surface sensitivity, as is demonstrated by diffusion measurements in model membranes with an excess of free dye in solution. All SA-FCS measurements are directly compared to standard confocal FCS, demonstrating a clear advantage of SA-FCS especially for diffusion measurements in membranes. We present an extensive theoretical framework which allows for accurate and quantitative evaluation of the SA-FCS correlation curves.

Key Words: FCS, SAF, TIRF, diffusion, membranes




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J. Ries, E. P. Petrov, and P. Schwille
Total Internal Reflection Fluorescence Correlation Spectroscopy: Effects of Lateral Diffusion and Surface-Generated Fluorescence
Biophys. J., July 1, 2008; 95(1): 390 - 399.
[Abstract] [Full Text] [PDF]


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Copyright © 2007 by the Biophysical Society.