Fluorescence Microscopy Imaging in Biomedical Sciences - Biomedical Optical Imaging Technologies: Design and Applications - page 97

Fig. 3.7 Comparison of the two measured instrument response functions (IRFs) of the TPE-
TCSPC FLIM system. Both IRFs were measured through collecting the second-harmonic
generation (SHG) signals emitted from urea crystals. At the 880-nm excitation wavelength, the
440-nm SHG emission signals were collected using a 460/50-nm emission filter. Under the 940-nm
excitation wavelength, the 470-nm SHG signals were collected using a 480/40-nm emission filter.
The two IRFs ( solid - the IRF measured at excitation 880 nm, emission 440 nm vs. dashed -
the IRF measured at excitation 940 nm, emission 470 nm) are almost overlaid with each other,
confirming that the IRF did not change for using the two different combinations of the TPE
wavelengths and the emission filters (Adapted from [ 47 ])

factors may influence the lifetime of the standard fluorophore. Figure 3.8 shows the
results of the calibrations of our TPE-TCSPC FLIM system with two fluorescence
lifetime standards: (1) coumarin 6 dissolved in ethanol has a peak absorption and
emission at 460 and 505 nm, and its fluorescence lifetime is 2:5 ns; (2) fluorescein
dissolved in phosphate buffer pH 7.5 has maximum absorption and emission at
495 and 517 nm, and its fluorescence lifetime is 4:0 ns (see http://www.iss.com/
resources/reference/data tables/LifetimeDataFluorophores.html ) .

3.4.5 Verifying the TPE-TCSPC FLIM System for FRET

Studies Using FRET Standards

As mentioned above, the FRET standard approach provides a quantitative way to
evaluate a FRET method. The utility of the TPE-TCSPC FLIM system for FRET
studies was assessed using four FP-based FRET-standard constructs expressed in
living cells [ 96 , 107 ]: C5V, C17V, C32V where C (Cerulean) and V (Venus) are
tethered by either a 5, 7, or 32 amino acid linker [ 107 ], and CTV where C and V are
separated by a 229 amino acid linker encoding a TRAF domain [ 96 ]. As shown in
Fig. 3.9 , these FRET-standard decay profiles were clearly distinguished in the TPE-
TCSPC FLIM-FRET measurements, demonstrating a shorter fluorescence lifetime
of Cerulean obtained from CTV to C32V to C17V to C5V cells and an increase