Minimization of detection volume by surface-plasmon-coupled emission.
We report theoretical predictions and experimental observations of the reduced detection volume with the use of surface-plasmon-coupled emission (SPCE). 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 direct excitation of the sample (reverse Kretschmann excitation) the detection volume is restricted only by the distance-dependent coupling of the excitation to the surface plasmons. However, with the excitation through the glass prism at surface plasmon resonance angle (Kretschmann configuration), the detection volume is a 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 10nm). The height of the detected volume size is 40-70nm, depending on the orientation of the excited dipoles. We show that, by using the Kretschmann configuration in a microscope with a high-numerical-aperture objective (1.45) together with confocal detection, the detection volume can be reduced to 1-2attoL. The strong dependence of the coupling to the surface plasmons on the orientation of excited dipoles can be used to study the small conformational changes of macromolecules.
Gryczynski
Z
Z
Department of Molecular Biology and Immunology, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107, USA.
Borejdo
J
J
Calander
N
N
Matveeva
E G
EG
Gryczynski
I
I
D000818:Animals; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D020349:Surface Plasmon Resonance / Q000295:instrumentation / Q000379:methods*
D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D011506:Proteins