From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells - Progress in Molecular Biology and Translational Science - page 166

spectrum, lifetime, anisotropy, and so on. In the particular case of biosensor
application, the most popular technique is called the “sensitized emission,”
which consists in acquiring the fluorescence emitted by the donor only and
the acceptor only, after donor excitation. However, several other
techniques offer very interesting alternatives for quantifying the molecular
activity, for example, (i) spectral imaging, which consists of excitation at
one wavelength and measurement of the whole emission spectrum, and (ii)
FLIM for measuring the lifetime changes of donor fluorescent proteins.
Each of these techniques has its own advantages and drawbacks. Using
two extreme examples, sensitized emission is the simplest method and
can be performed on nearly all conventional microscopes, but it requires
great care with regard to biological references; FLIM, on the other hand,
needs tricky instrumentation but can yield unambiguous measurements of
FRET efficiency. In the following section, systems needed to perform reli-
able biosensor imaging experiments are therefore described.
4.1. Intensity-based approaches
The most intuitive and easy methods to perform FRET measurements are
based on fluorescence intensity. The technique discussed now consists in
imaging the sensitized emission, that is, the fraction of acceptor emission in-
duced by the nonradiative energy transfer from the donor molecule. These
measurements can be based on either ratiometric or spectral imaging.
4.1.1 Ratiometric approach
FRET measurements using biosensors are usually performed by acquiring
the fluorescence emitted by the donor and the acceptor. The resulting data
are usually represented by the ratio of these fluorescence measurements after
appropriate corrections. The classical ratiometric approach, which is based
on Equation (5.14) , consists in measuring at least three channels: (i) excita-
tion and observation of the donor ( I donor ); (ii) excitation of the donor and
observation of the acceptor ( I FRET ); and (iii) excitation and observation
of the acceptor ( I acceptor ). I acceptor is needed to compensate for donor and ac-
ceptor concentration differences, thus accounting for bleed-through and
nonspecific excitation. 48 In the case of biosensor measurements performed
on living cells, the donor and acceptor amounts are identical and the last
channel acquisition is useless. In this particular case, only (i) and (ii) are re-
quired and, as will be seen in Section 5 , correction by acceptor channel will
even decrease the signal-to-noise ratio of the measured signal. This exper-
imental condition is formally similar to the well-described conditions used to