title: Fluorescence lifetime endoscopy using TCSPC for the measurement of FRET in live cells.
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 microm and 10 microm 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.
authors.affiliation: King's College London, The Richard Dimbleby Department of Cancer Studies, Division of Cancer, Guy's Medical School Campus, SE1 1UL, London, United Kingdom.
authors.forename: Gilbert O
authors.initials: GO
authors.lastname: Fruhwirth
authors.forename: Simon
authors.initials: S
authors.lastname: Ameer-Beg
authors.forename: Richard
authors.initials: R
authors.lastname: Cook
authors.forename: Timothy
authors.initials: T
authors.lastname: Watson
authors.forename: Tony
authors.initials: T
authors.lastname: Ng
authors.forename: Frederic
authors.initials: F
authors.lastname: Festy
mesh_terms: D019723:Endoscopes; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005336:Fiber Optic Technology / Q000295:instrumentation*; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation*; D007089:Image Enhancement / Q000295:instrumentation*; D008904:Miniaturization; D025941:Protein Interaction Mapping / Q000295:instrumentation*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity