Minimization of detection volume by surface plasmon-coupled emission

Surface plasmon-coupled emission (SPCE) has been used to reduce the detection volume in fluorescence measurements. The effective fluorescence volume (detection volume) in SPCE experiments depends on two near-field factors: the depth of evanescent wave excitation and a distance-dependent coupling of excited fluorophores to the surface plasmons. With the excitation through the glass prism at SPR angle (Kretschmann configuration), the detection volume is a composition (product) of evanescent wave penetration depth and distance-dependent coupling. In addition, the detection volume is further reduced by a metal quenching of excited fluorophores at a close proximity (below 10 nm). The height of the detected volume size is 40-70 nm, depending on the orientation of the excited dipoles. We show that using Kretchmann configuration in a microscope with high numerical aperture objective (1.45) together with confocal detection, the detection volume can be reduced to 1-2 attoL, which is necessary to observe a single cross-bridge in the muscle. The strong dependence of the coupling to the surface plasmons on the orientation of excited dipoles can be also used to study the small conformational changes of macromolecules.