From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells - Progress in Molecular Biology and Translational Science - page 201

the kinase of interest. The photoinduced effect arising from this high-energy
laser excitation must, however, be controlled. 71
For FLIM, the acquisition time strongly depends on the technique used.
For FD FLIM measurements using our setup, the exposure time of the do-
nor is between 100 and 300 ms with a laser power P average ¼

4 mW sent to a

spinning disk system equipped with a 63 1.4). To

achieve optimal compromise between the number of phase images and
the accuracy of lifetime determination, 12 phase-shifted images are chosen.
Moreover, achieving a good signal-to-noise ratio often requires averaging
each images three times. From these constraints, the overall exposure time
for one lifetime image can be between 3 s to almost 10 s. For time-resolved
lifetime measurements with our system, a field scan takes about 5 min (still
with an average laser power of a fewmilliwatts) and will require data binning
to achieve a precise lifetime map whereas the measurements per pixel only
takes few tens of milliseconds.
The choice of imaging technique thus depends on (i) the time resolution,
(ii) the number of images needed, and (iii) the spatial resolution. For exam-
ple, TCSPC can be used to obtain a precise image of the subcellular local-
ization of kinase activity before and after the activation of the biological
signal, while ratiometric measurement or phase and modulation technique
will be preferred to achieve a precise estimate of kinase activity kinetics over
time. The use of a biosensor targeted to subcellular compartment measured
by ratio imaging can also be a solution to achieve precise activity localization
without compromising acquisition speed.
In conclusion, we suggest avoiding any unnecessary exposure of
fluorophores, especially the donor, in FLIM experiments; moreover, pho-
tobleaching of the CFP will occur faster than for YFP variants. For example
(and if it is possible), find the focus using transmitted light rather than a mer-
cury lamp. Because of the numerous possible photoinduced artifacts, nega-
tive controls are indispensable.

oil objective (NA ¼

7.2.5 Negative control
Sensing applications require the creation of a control biosensor. Concerning
KAR, the phosphorylable residue within the substrate (serine, threonine, or
tyrosine) may be mutated to a nonphosphorylable amino acid such as glycine
or alanine. Expressed in cells, it should not produce a FRET signal in re-
sponse to the stimulation of the kinase of interest. It will therefore serve
as negative control and provide the basal level of FRET signal in living cells.
An example of such an experiment is presented in Fig. 5.25 . There is,