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.
Highlights
One of modern medicine’s most rapidly expanding non-serological diagnostic technologies has arisen from the development of macroscopic reflection instruments in the 1960’s, allowing wide field imaging and tissue biopsy via “non-invasive” endoscopy [1,2]
The recent advent of a clinically licensed intensity-based fluorescence microendoscopic instrument has offered the prospect of real-time cellular resolution imaging
We developed a new instrument combining remote fiber endoscopic cellular-resolution imaging with time-correlated single-photon counting (TCSPC)-FLIM technology to interrogate and discriminate mixed fluorochrome labeled beads and expressible GFP/TagRFP tags within live cells
Summary
One of modern medicine’s most rapidly expanding non-serological diagnostic technologies has arisen from the development of macroscopic reflection instruments in the 1960’s, allowing wide field imaging and tissue biopsy via “non-invasive” endoscopy [1,2]. There is a huge clinical pressure to develop high resolution, in situ optodiagnostic micro-endoscopic technologies with the potential to provide immediate tissue characterization and diagnostic information This will allow more extensive tissue sampling with little or no consequential tissue morbidity and in clinical practice more timely implementation of appropriate therapies [3]. Recent advances in fluorescence lifetime imaging have enabled numerous studies of protein-interaction and physiological monitoring at the microscopic level [5] This minirevolution of studies was catalyzed first by the improvement in gated and modulated image intensifier technology in wide-field microscopy and latterly by novel light sources [6] and modern signal processing electronics for time-correlated single-photon counting (TCSPC) [7] or frequency domain routes [8,9].
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