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Originally published as Biophys J. BioFAST on September 7, 2007.
doi:10.1529/biophysj.107.115998
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Biophysical Journal 94:221-229 (2008)
© 2008 The Biophysical Society

Supercritical Angle Fluorescence Correlation Spectroscopy

Jonas Ries *, Thomas Ruckstuhl {dagger}, Dorinel Verdes {dagger} and Petra Schwille *

* Biotechnologisches Zentrum, Technical University of Dresden, Dresden, Germany; and {dagger} Institute of Physical Chemistry, University of Zürich, Zürich, Switzerland

Correspondence: Address reprint requests to Petra Schwille, Tel.: 49-351-463-40328; E-mail: schwille{at}biotec.tu-dresden.de.

We explore the potential of a supercritical angle (SA) 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 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 that allows for accurate and quantitative evaluation of the SA-FCS correlation curves.




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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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