New surface plasmons approach to Single Molecule Detection (SMD) and Fluorescence Correlation Spectroscopy (FCS)

Zygmunt Gryczynski, Ignacy Gryczynski, E. G. Matveeva, N. Calander, R. Grygorczyk, I. Akopova, S. Bharill, P. Muthu, S. Klidgar, Julian Borejdo

Research output: Chapter in Book/Report/Conference proceedingConference contribution

4 Scopus citations

Abstract

We report new approach to Fluorescence Correlation Spectroscopy (FCS) and Single Molecule Detection (SMD) based on Surface Plasmon-Coupled Emission (SPCE) technology. The use of SPCE offers significant reduction of fluorescence volume (detection volume) reduction decreasing background contribution. Fluorophore interaction with surface plasmons increases the rate of photon detection and makes fluorescence very sensitive to change in a position of emitting molecule. The effective thickness of the fluorescence volume in SPCE experiments depends on two factors: the depth of evanescent wave excitation and a distance-dependent coupling of excited fluorophores to the surface plasmons. The excitation with the laser beam at Surface Plasmon Resonance (SPR) angle (Kretschmann configuration) through the high numerical aperture objective makes observation volume very shallow below 100 nm. The layer thickness is further reduced by the metal quenching of excited fluorophores immediately close to the interface (∼10 nm). The fluorescence light is emitted through the metal film only at the SPCE angle. Any fluorescence occurring at the distances greater than the coupling distance is effectively reflected (∼92%) by the metal film and not transmitted to the objective. The thickness of the detected volume can be 20-50 nm, depending on the prism dielectric constants and orientation of the excited dipoles. In addition the signal is very sensitive to the change in fluorophore position and orientation. Such strong dependence of the coupling to the surface plasmons on the orientation of excited dipoles opens new possibilities to study conformational changes of macromolecules in real time.

Original languageEnglish
Title of host publicationUltrasensitive and Single-Molecule Detection Technologies II
DOIs
StatePublished - 30 Apr 2007
EventUltrasensitive and Single-Molecule Detection Technologies II - San Jose, CA, United States
Duration: 20 Jan 200723 Jan 2007

Publication series

NameProgress in Biomedical Optics and Imaging - Proceedings of SPIE
Volume6444
ISSN (Print)1605-7422

Other

OtherUltrasensitive and Single-Molecule Detection Technologies II
CountryUnited States
CitySan Jose, CA
Period20/01/0723/01/07

Keywords

  • Fluorescence
  • Fluorescence correlation spectroscopy
  • Macromolecular conformational changes
  • Minimized detection volume
  • Surface plasmon-coupled emission

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    Gryczynski, Z., Gryczynski, I., Matveeva, E. G., Calander, N., Grygorczyk, R., Akopova, I., Bharill, S., Muthu, P., Klidgar, S., & Borejdo, J. (2007). New surface plasmons approach to Single Molecule Detection (SMD) and Fluorescence Correlation Spectroscopy (FCS). In Ultrasensitive and Single-Molecule Detection Technologies II [64440G] (Progress in Biomedical Optics and Imaging - Proceedings of SPIE; Vol. 6444). https://doi.org/10.1117/12.715268