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

Fluorescence Lifetime Endoscopy Using TCSPC for the Measurement of FRET in Live Cells

Free PMC article

Fluorescence Lifetime Endoscopy Using TCSPC for the Measurement of FRET in Live Cells

Gilbert O Fruhwirth et al. Opt Express. .
Free PMC article


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.


Fig. 1
Fig. 1
Schematic drawing of the experimental setup. (A) Main components of the setup are shown including a magnified reflection image of the front of the fiber bundle. The fiber cores have a diameter of 2.28 ± 0.48 µm and are spaced 2.80 ± 0.21 µm apart, leading to an optical resolution of about 5 µm. (B) Illustration of how the fiber bundle is coupled to the objective. The laser beam is scanned over the polished fiber bundle, which is aligned precisely with the focal plane.
Fig. 2
Fig. 2
Comparison of wide-field intensity and single-photon TCSPC FLIM measurements of small beads using either fluorescence lifetime microscopy or endoscopy through a coherent fiber bundle. (A) 10 µm and 4 µm microspheres were immersed alone (top and middle panels, respectively) or as a mixture in a clear 3D matrix and the bottom layer was imaged using a 20-fold objective (0.5 NA, 2.1 mm WD). From the single photon intensity FLIM images the fluorescence lifetime maps were calculated by applying mono-exponential fitting. The right column shows the corresponding fluorescence lifetime histograms. (B) The same samples were used for fluorescence lifetime endoscopy through a coherent fiber bundle of 2 m length and the data are shown as described for (A). Scale bars are 50 µm.
Fig. 3
Fig. 3
Fluorescence lifetime endoscopy of living mammalian cells. Wide-field and single-photon TCSPC images of cells expressing CXCR4-GFP and CXCR4-RFP together or CXCR4-GFP alone (control) are shown. The cells expressing both receptors show FRET between GFP and RFP due to receptor dimerization. Fluorescence lifetime maps are calculated from single-photon intensity images and the corresponding histograms are shown next to the fluorescence lifetime maps. (A) Images acquired in the microscopic mode. (B) Images acquired in the endoscopy mode. Scale bars are 20µm.

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