From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells - Progress in Molecular Biology and Translational Science - page 167

Table 5.2 Example of the adapted filter set for the CFP/YFP FRET pair 48
Channel Excitation filter Excitation dichroic Emission filter

I donor 420/20 450 475/40

I FRET 420/20 450 535/25

image calcium with ratiometric indicators, except that calcium probes such
as fura-2 show a ratiometric change for two different excitation wavelengths
while biosensors show a ratiometric change of the fluorescence emission for
one excitation wavelength.
One major advantage of this technique is that it requires only two wide-
field or confocal images, allowing for high-speed acquisitions of fluores-
cence images with systems equipped with appropriate filter sets available
in most imaging core facilities ( Table 5.2 ).
Special care must, however, be taken to limit the delay between the
acquisition of I donor and I FRET to avoid artifacts due to cell movement be-
tween two image acquisitions (see Section 7 of this chapter). The traditional
system needs a filter cube change and thus a delay of several hundreds of mil-
liseconds between image acquisitions is possible, which is not negligible
compared to the movement times of cells or organelles. Instead of changing
the fluorescence cube, which may worsen image registration, a faster solu-
tion is the use of a filter wheel between the microscope and the camera,
which allows changes to be made in the emission filter in less than
100 ms. Solutions have also been developed to perform simultaneous acqui-
sitions of both channels, as described in Fig. 5.11 . 49-51
These wide-field configurations allow fast acquisition of both channels
but they lack optical sectioning capability. Confocal microscopes can also
be used in the same way, with excitation of the donor and simultaneous col-
lection of the fluorescence emitted by donor and acceptor molecules. This
allows for a three-dimensional localization of the biosensor response, and
video-rate confocal microscopes can be used when a high spatial resolution
is needed, as shown by the early use of biochemical biosensors. 52 The use of
confocal microscopes also opens the way to more complex acquisition pro-
cedures such as spectral imaging.

4.1.2 Spectral imaging
Most of the latest confocal microscopes are designed for spectral imaging
either in the sequential mode or, most interestingly, for biosensor imaging in
the simultaneous mode. 53 Indeed, for FRET applications, spectral imaging is