Journal
ANALYTICAL BIOCHEMISTRY
Volume 356, Issue 1, Pages 125-131Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2006.05.007
Keywords
surface-plasmon-coupled emission; minimized detection volume; attoliter; fluorescence microscope
Funding
- NCI NIH HHS [CA-114460] Funding Source: Medline
- NIAMS NIH HHS [AR-048622] Funding Source: Medline
- NIBIB NIH HHS [EB-1690] Funding Source: Medline
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We report theoretical predictions and experimental observations of the reduced detection volume with the use of surface-plasmoncoupled 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 10 nm). The height of the detected volume size is 40-70 nm, depending on the orientation of the excited dipoles. We show that, by using the Kretschmarm configuration in a microscope with a high-numerical-aperture objective (1.45) together with confocal detection, the detection volume can be reduced to 1-2 attoL. 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. (c) 2006 Elsevier Inc. All rights reserved.
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