Fluorescence correlation spectroscopy in a reverse Kretchmann surface plasmon assisted microscope

Optics Express
N CalanderJulian Borejdo

Abstract

Fluorescence Correlation Spectroscopy (FCS) demands a high rate of photon detection per molecule, low background, and large fluctuations of fluorescence associated with translational motion. The new approach presented here, Surface Plasmon Assisted Microscope (SPAM), meets these requirements by drastically limiting the observation volume. In this method, the observational layer is made so thin that fluctuations are mostly due to the axial motion of molecules. This is conveniently realized by placing a sample on a thin metal film and illuminating it with a laser beam through an aqueous medium. The excited fluorophores close to the surface couple (via near-field interactions) to surface plasmons in the metal. Propagated surface plasmons decouple on opposite side of the metal film as a far-field radiation and emit in directional manner. Fluorescence is collected with a high Numerical Aperture objective. A confocal aperture inserted in its conjugate image plane reduces lateral dimensions of the detection volume to a diffraction limit. The thickness of the detection layer is reduced further by metal quenching of excited fluorophores at a close proximity (about 30 nm) to the surface. We used a suspension of fluorescent microspheres t...Continue Reading

References

Oct 29, 2003·Biophysical Journal·Petra Schwille
Mar 24, 2004·Optics Letters·Thomas Ruckstuhl, Stefan Seeger
Aug 10, 2004·Biophysical Journal·Dieter Braun, Peter Fromherz
Jun 13, 2006·Analytical Biochemistry·Z GryczynskiI Gryczynski
Oct 31, 2005·Optics Express·Jörg Enderlein, Thomas Ruckstuhl
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Nov 22, 2005·Thin Solid Films·Ignacy GryczynskiZygmunt Gryczynski

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Citations

Jan 13, 2015·The Journal of Physical Chemistry. B·Qian LiuYao-Qun Li
Oct 4, 2012·The Journal of Physical Chemistry Letters·Sharmistha Dutta ChoudhuryJoseph R Lakowicz
Dec 10, 2008·Optics Express·Laura C EstradaOscar E Martínez
Apr 18, 2020·Analytical and Bioanalytical Chemistry·Min ChenYao-Qun Li
Jun 24, 2018·Journal of Biomedical Optics·Julian BorejdoIgnacy Gryczynski

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