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

Opt Express. 2010 May 24;18(11):11148-58. doi: 10.1364/OE.18.011148.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Endoscopes*
  • Equipment Design
  • Equipment Failure Analysis
  • Fiber Optic Technology / instrumentation*
  • Fluorescence Resonance Energy Transfer / instrumentation*
  • Image Enhancement / instrumentation*
  • Miniaturization
  • Protein Interaction Mapping / instrumentation*
  • Reproducibility of Results
  • Sensitivity and Specificity