Fluorescence Microscopy Imaging in Biomedical Sciences - Biomedical Optical Imaging Technologies: Design and Applications - page 99

Fig. 3.9 Verification of the TPE-TCSPC FLIM-FRET approach using FRET-standard constructs -
C5V, C17V, C32V, and CTV (see Sect. 3.4.5 ). The Cerulean fluorescence lifetimes in live cells
expressing Cerulean alone (C) or a FRET-standard construct (CTV, C32V, C17V, or C5V) were
measured using the TPE-TCSPC FLIM system. All acquired data were analyzed with single (for
C and CTV) or double (for C32V, C17V, and C5V) exponential fittings. For each construct, the
representative decay profile (at one pixel of each cell) and the lifetime distribution (in each cell)
are shown, clearly demonstrating a faster decay (a shorter lifetime) from C to C5V, as expected
from the designed linker systems (Adapted from [ 43 ])
was preferentially bound to satellite DNA-repeat sequences located in regions of
centromeric heterochromatin [ 180 , 181 ]. Standard recombinant DNA methods were
used to fuse the sequences that code for either Cerulean or Venus into the reading
frame of the sequence encoding the rat C=EBP' bZip domain, starting with the
methionine at position 237 [ 182 ]. When the C=EBP' bZip domain is expressed
as a fusion to the FPs in cells of mouse origin, such as the pituitary GHFT1 cells
used here, it is localized to the well-defined regions of centromeric heterochromatin
in the cell nucleus [ 78 , 183 ]. As shown in Fig. 3.10 , the TPE-TCSPC FLIM-FRET
approach is a perfect tool to demonstrate the homodimerization of C=EBP' in live
mouse pituitary cell nuclei based on the detection of FRET between Cerulean and
Venus tagged to C=EBP'.