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Published on: December 11, 2013
Minimization of detection volume by surface-plasmon-coupled emission
Z Gryczynski1, J Borejdo, N Calander
1Department of Molecular Biology and Immunology, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107, USA.
Analytical Biochemistry
|June 13, 2006
Summary
Surface-plasmon-coupled emission (SPCE) significantly reduces detection volume using near-field effects. This technique, particularly the Kretschmann configuration, enables ultra-small detection volumes for studying molecular changes.
Area of Science:
- Optics and Photonics
- Biophysics
- Materials Science
Background:
- Surface-plasmon-coupled emission (SPCE) relies on near-field interactions.
- The detection volume in SPCE is influenced by evanescent wave depth and fluorophore coupling to surface plasmons.
Purpose of the Study:
- To theoretically predict and experimentally observe the reduced detection volume in SPCE.
- To investigate factors limiting detection volume, including excitation methods and metal quenching.
- To demonstrate ultra-small detection volumes for macromolecular studies.
Main Methods:
- Utilized surface-plasmon-coupled emission (SPCE) with both reverse Kretschmann and Kretschmann excitation configurations.
- Employed a high-numerical-aperture objective (1.45) and confocal detection microscopy.
- Investigated the influence of fluorophore proximity to metal surfaces and dipole orientation.
Main Results:
- SPCE, especially the Kretschmann configuration, drastically reduces the effective fluorescence detection volume.
- Detection volume is limited by evanescent wave penetration, surface plasmon coupling, and metal quenching.
- Achieved detection volumes as small as 1-2 attoLiters (10^-18 L) with optimized microscopy.
- Observed a detection volume height of 40-70 nm, dependent on excited dipole orientation.
Conclusions:
- SPCE offers a powerful method for achieving ultra-small detection volumes in fluorescence microscopy.
- The orientation-dependent coupling to surface plasmons can be exploited to probe conformational changes in macromolecules.
- This technique holds promise for high-resolution biophysical studies.
