Fluorescent Protein-Based Biosensors and Their Clinical Applications - Progress in Molecular Biology and Translational Science - page 328

50
100 ¼

E ¼ 1 0 : 5 : ½ 8 : 12

Therefore, 50% of the excited energy of CFP is transferred to YFP in the
presence of A-B interaction. However, an increase in donor fluorescence
after photobleaching can occur independently of the association between
A and B. In addition, this method can therefore be executed only once
because of the destructive nature of the photobleaching necessary to obtain E .
It is important to note that, in the aforementioned method, only the in-
tensity of donor fluorescence is measured. Because acceptor molecules (such as
YFP) not only absorb but also emit light in most FRET experiments, FRET
can also be quantified by measuring the sensitized emission from the acceptor.
When the sample is excited at the donor excitation wavelengths, fluorescence
intensities from the acceptor and donor molecules are increased and decreased,
respectively, in a manner that depends on the efficacy of energy transfer.
Therefore, E can be estimated using the ratio of fluorescence intensities emit-
ted at donor versus acceptor emission wavelengths. 90 This dual-emission ratio
method can repeatedly measure and reliably detect changes in FRET, regard-
less of the background FRET levels. However, the dual-emission ratio
method is not as quantitative as donor fluorescence measurement because
the ratio is affected by unavoidable factors deriving from optical instruments
(specification of the used filters or detectors). 60 Thus, the acceptor/donor
emission ratio can be used as an index of the degree of FRET.
The emission ratio, specifically the acceptor emission obtained by excita-
tion of the donor, is also affected by factors other than the FRET efficiency. In
the case of CFP-YFP FRET, excitation of the donor molecule (440 nm for
CFP) also excites YFP, albeit less efficiently. Also, the CFP emission leaks into
the FRET channel. Therefore, stoichiometric alterations in the amounts of
donor and acceptor result in observed intensities of sensitized emission even
in the absence of changes in FRET efficiency. To overcome this issue, re-
searchers who wish to obtain reliable FRET indices must perform spectral
unmixing. As described in Section 2.5 , cells expressing only the donor or
the acceptor are imaged through three sets of optical filters (donor
excitation-donor emission, donor excitation-acceptor emission, and acceptor
excitation-acceptor emission) under the same imaging conditions used for
FRET observation. This experiment provides slopes that represent the
bleed-through of CFP and YFP into the FRET channel (i.e.,

CFP bt YFP; bt, bleed-through). In the case of FRET

with CFP and YFP, the level of cross talk between the donor and the acceptor

¼ a CFP; YFP bt ¼ b