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Surface plasmon-coupled emission (SPCE) is a phenomenon whereby the light emitted from a fluorescent molecule can couple into the surface plasmon of an adjacent metal layer, resulting in highly directional emission in the region of the surface plasmon resonance (SPR) angle. In addition to high directionality of emission, SPCE has the added advantage of surface selectivity in that the coupling diminishes with increasing distance from the surface. This effect can be exploited in bioassays whereby a fluorescing background from the sample can be suppressed. We have investigated, both theoretically and experimentally, the SPCE effect for a Cy5-spacer-Ag layer system. Both the angular dependence of emission and the dependence of SPCE emission intensity on Cy5-metal separation were investigated. It is demonstrated that SPCE leads to lower total fluorescence signal than that obtained in the absence of a metal layer. This is the first experimental verification of the reduction in SPCE intensity compared to the metal-free case. Our results are in a good agreement with theoretical models. The validation of the theoretical model provides a basis for optimizing biosensor platform performance, particularly in the context of the advantages offered by SPCE of highly directional emission and surface selectivity.

We report a new two-channel fluorescence microscopy technique for surface-generated fluorescence. The realized fluorescence microscope allows high resolution imaging of aqueous samples. The core element of the instrument is a parabolic mirror objective that is used to collect the fluorescence at large surface angles above the critical angle of the waterglass interface. An aspheric lens, incorporated into the solid parabolic element, is used for diffraction-limited laser focusing and for collecting fluorescence at low angles with respect to the optical axis. By separated collection of the fluorescence emitted into supercritical and subcritical angles, two detection volumes strongly differing in their axial resolution are generated at the surface of a glass cover slip. The collection of supercritical angle fluorescence (SAF) results in a strict surface confinement of the detection volume, whereas collecting below the critical angle allows gathering the fluorescence emitted several microns deep inside the sample. Consequently, the signals from surface-bound and unbound diffusing fluorescent molecules can be obtained simultaneously.