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Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 15, Iss. 11 — May. 28, 2007
  • pp: 6934–6945

Imaging of protein cluster sizes by means of confocal time-gated fluorescence anisotropy microscopy

Arjen N. Bader, Erik G. Hofman, Paul M. P. van Bergen en Henegouwen, and Hans C. Gerritsen  »View Author Affiliations


Optics Express, Vol. 15, Issue 11, pp. 6934-6945 (2007)
http://dx.doi.org/10.1364/OE.15.006934


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Abstract

A time-resolved fluorescence anisotropy imaging method for studying nanoscale clustering of proteins or lipids was developed and evaluated. It is based on FRET between the identical fluorophores (homo-FRET), which results in a rapid depolarization of the fluorescence. The method employs the time-resolved fluorescence anisotropy decays recorded in a confocal microscope equipped with pulsed excitation and time-gated detection. From the decay the limiting anisotropy rinf was derived, which is a direct measure for the number of fluorophores per cluster. The method was evaluated by imaging GPI-GFP, a lipid raft marker. Small clusters were observed in the plasma membrane while the cytoplasm and the Golgi contained predominantly monomers.

© 2007 Optical Society of America

OCIS Codes
(170.1530) Medical optics and biotechnology : Cell analysis
(180.1790) Microscopy : Confocal microscopy
(180.2520) Microscopy : Fluorescence microscopy
(260.2160) Physical optics : Energy transfer
(260.5430) Physical optics : Polarization

ToC Category:
Microscopy

History
Original Manuscript: April 6, 2007
Revised Manuscript: May 15, 2007
Manuscript Accepted: May 15, 2007
Published: May 21, 2007

Virtual Issues
Vol. 2, Iss. 6 Virtual Journal for Biomedical Optics

Citation
Arjen N. Bader, Erik G. Hofman, Paul M. P. van Bergen en Henegouwen, and Hans C. Gerritsen, "Imaging of protein cluster sizes by means of confocal time-gated fluorescence anisotropy microscopy," Opt. Express 15, 6934-6945 (2007)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-15-11-6934


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