Fluorescence Microscopy Imaging in Biomedical Sciences - Biomedical Optical Imaging Technologies: Design and Applications - page 98

Fig. 3.8 Calibrations of the TPE-TCSPC FLIM system with two fluorescence lifetime standards -
coumarin 6 dissolved in ethanol and fluorescein dissolved in phosphate buffer pH 7.5. For both
coumarin 6 and fluorescein, the single exponential decay model was applied to fit the acquired
data; the representative raw decay traces and corresponding fitting curves are shown together
with the weighted least squares ( 2 ) and residues determined by comparing the raw and fitted
data, indicating good fits. The fluorescence lifetimes of coumarin 6 (2.51 ns) and fluorescein
(3.99 ns) obtained through fitting closely matched their reference values (coumarin 6 - 2.5 ns and
fluorescein - 4.0 ns)

in FRET efficiency (E) in the same direction. The Es of these FRET-standard
constructs were determined using the TPE-TCSPC FLIM-FRET method based on
Eq. 3.3 , where the unquenched Cerulean lifetime ( D ) was obtained through the
single exponential fitting of the decay data acquired from cells expressing Cerulean
alone; the quenched Cerulean lifetime for each FRET-standard construct ( DA )was
estimated by fitting its decay data into a single or double exponential model; the
resultant Es matched with those measured by other FRET methods including FD
FLIM-FRET, spectral FRET, and filter-based confocal FRET [ 100 , 179 ].

3.4.6 Detecting Protein-Protein Interactions in Living Cells

with TPE-TCSPC FLIM-FRET

The biological model used here is the basic region-leucine zipper (bZip) domain
of the C=EBP' transcription factor. The bZip family proteins form obligate
dimers through their leucine-zipper domains, which positions the basic region
residues for binding to specific DNA elements. Immunocytochemical staining of
differentiated mouse adipocyte cells showed that the endogenous C=EBP' protein