From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells - Progress in Molecular Biology and Translational Science - page 174

where c is a constant that is dependent on the fluorophore's properties. This
constant, which is just an offset modification of the ratio R , does not give any
supplementary information.
For a single-chain biosensor, the ratio R is then fully described by the
ratio of the fluorescence intensities, I FRET / I Donor . Consequently, the third
fluorescence image I Acceptor (see Eq. 5.27 ) is not useful; this additional image
acquisition will just increase the complexity of instrumentation, the time of
acquisition (and thus the related movement artifacts), and the noise propa-
gation in the ratio calculation.
Even though the expression of the ratio is extremely simple, the fact must
be emphasized that obtaining reliable R values requires special care and
numerous corrections, which have been exhaustively detailed elsewhere. 68,69
Briefly, we need to compensate for instrumental artifacts (camera
offset subtraction, shading or flat-field correction for the nonuniformity of
the illumination, correction for misalignments between two fluorescence
images due to chromatic aberration) and for chemical and biological
artifacts (photobleaching correction for taking into account the different
donor and acceptor photobleach rates 70 , optional autofluorescence
subtraction, correction for cell movement, or deformation between
acquisition time).
Calculation of the corrected ratio R from acquired fluorescence images
can be performed instantaneously pixel by pixel, and the calculated R values
can be displayed with pseudocolor superimposed on the fluorescence
intensity image in real time, making it possible to follow the biosensor
activity 71-73 (see Fig. 5.16 ). Ideally, for a sensor binding to a single
molecule (such as fura-2), the ligand concentration [L] is directly related
to the ratio as 74

R min
R max

R

½/ L ½ 5 : 29

R

where R min and R max are, respectively, the minimum and maximum ratios.
However, regarding single-chain biosensors, it is usually not possible to have
access to these ratios. In this case, the normalized ratio D R/R to can be used to
facilitate the comparison between all cell responses (cf. Fig. 5.16 ). This nor-
malized ratio is defined by

D R
R t 0 ¼

j R R t 0 j

½ 5 : 30

R t 0