Fluorescence correlation spectroscopy in surface plasmon coupled emission microscope.

Study of dynamics of single molecules by Fluorescence Correlation Spectroscopy (FCS) requires that the rate of photon detection per molecule be high, that the background be low, and that there be a large change in fluorescent signal associated with change in a position of a molecule. FCS applied to microscopic Surface Plasmon Coupled Emission (SPCE) suggests a powerful method to meet those requirements. In this method, the observational volume is made shallow by placing a sample on a thin metal film and illuminating it with the laser beam at Surface Plasmon Resonance (SPR) angle through high numerical aperture objective. The illuminating light excites surface plasmons in the metal film that produce an evanescent wave on the aqueous side of the interface. The thickness of the detection volume is a product of evanescent wave penetration depth and distance-dependent fluorescence coupling to surface plasmons. It is further reduced by a metal quenching of excited fluorophores at a close proximity (below 10 nm) to a surface. The fluorescent light is emitted through the metal film only at an SPCE angle. Objective collects emitted light, and a confocal aperture inserted in its conjugate image plane reduces lateral dimensions of the detection volume to a fraction of a micrometer. By using diffusion of fluorescent microspheres, we show that SPCE-FCS is an efficient method to measure molecular diffusion and that on gold surface the height of the detection volume is ~35 nm.