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Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 18, Iss. 11 — May. 24, 2010
  • pp: 11148–11158

Fluorescence lifetime endoscopy using TCSPC for the measurement of FRET in live cells

Gilbert O. Fruhwirth, Simon Ameer-Beg, Richard Cook, Timothy Watson, Tony Ng, and Frederic Festy  »View Author Affiliations


Optics Express, Vol. 18, Issue 11, pp. 11148-11158 (2010)
http://dx.doi.org/10.1364/OE.18.011148


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Abstract

Development of remote imaging for diagnostic purposes has progressed dramatically since endoscopy began in the 1960’s. The recent advent of a clinically licensed intensity-based fluorescence micro-endoscopic instrument has offered the prospect of real-time cellular resolution imaging. However, interrogating protein-protein interactions deep inside living tissue requires precise fluorescence lifetime measurements to derive the Förster resonance energy transfer between two tagged fluorescent markers. We developed a new instrument combining remote fiber endoscopic cellular-resolution imaging with TCSPC-FLIM technology to interrogate and discriminate mixed fluorochrome labeled beads and expressible GFP/TagRFP tags within live cells. Endoscopic-FLIM (e-FLIM) data was validated by comparison with data acquired via conventional FLIM and e-FLIM was found to be accurate for both bright bead and dim live cell samples. The fiber based micro-endoscope allowed remote imaging of 4 µm and 10 µm beads within a thick Matrigel matrix with confident fluorophore discrimination using lifetime information. More importantly, this new technique enabled us to reliably measure protein-protein interactions in live cells embedded in a 3D matrix, as demonstrated by the dimerization of the fluorescent protein-tagged membrane receptor CXCR4. This cell-based application successfully demonstrated the suitability and great potential of this new technique for in vivo pre-clinical biomedical and possibly human clinical applications.

© 2010 OSA

OCIS Codes
(170.2150) Medical optics and biotechnology : Endoscopic imaging
(170.2520) Medical optics and biotechnology : Fluorescence microscopy

ToC Category:
Medical Optics and Biotechnology

History
Original Manuscript: February 3, 2010
Revised Manuscript: March 31, 2010
Manuscript Accepted: April 13, 2010
Published: May 12, 2010

Virtual Issues
Vol. 5, Iss. 10 Virtual Journal for Biomedical Optics

Citation
Gilbert O. Fruhwirth, Simon Ameer-Beg, Richard Cook, Timothy Watson, Tony Ng, and Frederic Festy, "Fluorescence lifetime endoscopy using TCSPC for the measurement of FRET in live cells," Opt. Express 18, 11148-11158 (2010)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-18-11-11148


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