From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells - Progress in Molecular Biology and Translational Science - page 173

Modulated laser source

Signal generator

Rotation axe

Microlenses disk

Pinhole disk

Figure 5.15 Scheme of a phase and modulation acquisition system adapted on a spin-
ning disk system to ensure fast fluorescence lifetime measurement with optical section-
ing capability.

I FRET a I Donor b I Acceptor

R ¼ ½ 5 : 27

I Donor

where I Acceptor is the fluorescence intensity measured with an acceptor emis-
sion filter after acceptor excitation, I Donor and I FRET are fluorescence inten-
sities measured, respectively, with the spectral bandpass of the donor and
acceptor after donor excitation, and a and b are the correction factors
for, respectively, the bleed-through of the donor into the acceptor emission
filter after donor excitation and the bleed-through of the acceptor into the
acceptor emission filter after donor excitation.
In the particular case of a single-chain biosensor, the expression (27) can
be simplified. Indeed, donor and acceptor concentrations are identical,
which implies that bleed-through correction factors a and b are linearly de-
pendent and that fluorescence intensities I Acceptor and I Donor are propor-
tional. Taking into account these simplifications, a straightforward

calculation leads to

I FRET

R ¼

I Donor c

½ 5 : 28