Journal
OPTICS EXPRESS
Volume 18, Issue 11, Pages 11148-11158Publisher
Optica Publishing Group
DOI: 10.1364/OE.18.011148
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Funding
- CR-UK
- EPSRC [C1519/A10331]
- Dimbleby Cancer Care
- NIHR [BS/DHCS/03/G121/55]
- Department of Health via the National Institute for Health Research (NIHR)
- King's College Hospital NHS Foundation Trust
- Medical Research Council Co-Operative Group [G0100152 ID 56891]
- UK Research Councils [GR/R87901/01]
- Guy's and St. Thomas Hospitals' Charitable Foundation
- Engineering and Physical Sciences Research Council [GR/R87901/01] Funding Source: researchfish
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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 Forster 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 mu m and 10 mu m 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. (C) 2010 Optical Society of America
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