New surface plasmons approach to single molecule detection (SMD) and fluorescence correlation spectroscopy (FCS)

Abstract

We report new approach to Fluorescence Correlation Spectroscopy (FCS) and Single Molecule Detection (SMD) based on Surface Plasmon-Coupled Emission (SPCE) technology. The use of SPCE offers significant reduction of fluorescence volume (detection volume) reduction decreasing background contribution. Fluorophore interaction with surface plasmons increases the rate of photon detection and makes fluorescence very sensitive to change in a position of emitting molecule. The effective thickness of the fluorescence volume in SPCE experiments depends on two factors: the depth of evanescent wave excitation and a distance-dependent coupling of excited fluorophores to the surface plasmons. The excitation with the laser beam at Surface Plasmon Resonance (SPR) angle (Kretschmann configuration) through the high numerical aperture objective makes observation volume very shallow below 100 nm. The layer thickness is further reduced by the metal quenching of excited fluorophores immediately close to the interface (~10 nm). The fluorescence light is emitted through the metal film only at the SPCE angle. Any fluorescence occurring at the distances greater than the coupling distance is effectively reflected (~92%) by the metal film and not transmitted to the objective. The thickness of the detected volume can be 20-50 nm, depending on the prism dielectric constants and orientation of the excited dipoles. In addition the signal is very sensitive to the change in fluorophore position and orientation. Such strong dependence of the coupling to the surface plasmons on the orientation of excited dipoles opens new possibilities to study conformational changes of macromolecules in real time.