Fluorescence Microscopy Imaging in Biomedical Sciences - Biomedical Optical Imaging Technologies: Design and Applications - page 100

Fig. 3.10 Localization of homodimerization of CCAAT/enhancer-binding protein alpha
.C=EBP'/ in live mouse pituitary cell nuclei using TPE-TCSPC FLIM-FRET microscopy.
C=EBP' was tagged with either Cerulean (C) or Venus (V). The fluorescent lifetime decay kinetics
for the C C=EBP' (FRET

acceptor) were determined by fitting the measured decay data into a single or double exponential
decay model, respectively, with the measured instrument function (IRF). By applying a suitable
threshold, fittings were only applied to the pixels in regions of centromeric heterochromatin of
the cell nucleus. The comparison between the representative measured decay data points, fitting
curves, and lifetime images (overlaid with intensity) of the two cases clearly shows the C in cells
expressing “C

C=EBP' (FRET donor) in the absence and the presence of V

C=EBP'” decayed faster (or has a shorter lifetime) than that
in cells expressing C-C/EBP˛ alone, indicating the C attached to C/EBP˛ was quenched by the
V attached to C=EBP' due to FRET and further demonstrating the interaction between C- and
V-tagged C=EBP' (homodimerization of C=EBP') in regions of centromeric heterochromatin of
the cell nucleus (Adapted from [ 166 ])

C=EBP' C V

3.5 Conclusion and Outlook

Being the most rapidly expanding microscopy technique employed today, fluores-
cence microscopy will no doubt continue to remain on the central stage of the
life sciences. The various applications using fluorescence microscopy technologies
described in this chapter and many others not described here will continue to
increase in all directions of the life sciences, driven by the growing number of
biologists who routinely use these technologies. Many new advanced fluorescence
microscopy techniques will continue to be developed, driven by development of new
fluorescent probes, invention of new sophisticated microscope hardware and soft-
ware, and the need to solve more complicated biological questions. Although some
of the fluorescence microscopy techniques (e.g., FLIM described in Sect. 3.4 ) can be
somewhat challenging for biologists without physics background, they provide an
unprecedented level of information about functions, dynamics, and interactions of
proteins in cells under physiological conditions at a very high temporal and spatial