Fluorescence correlation spectroscopy in a reverse Kretchmann surface plasmon assisted microscope.

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 to show that FCS-SPAM is an efficient method to measure molecular diffusion.