From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells - Progress in Molecular Biology and Translational Science - page 172

has contributed to the simplification of the instrumentation and reduced the
cost of FD FLIM systems. 57-59
Measurement of the phase and modulation quantities can be performed
with two approaches: the heterodyne and the homodyne methods. The het-
erodyne method is the preferred approach for accurately estimating fluores-
cence lifetime components in cuvette experiments. It has also been
successfully applied in FLIM experiments in combination with scanning-
mode and single-channel detectors (usually with a gain-modulated photo-
multiplier 54 ). This method is thus compatible with laser scanning micro-
scopes (such as confocal and multiphoton microscopes), which offer high
three-dimensional spatial resolution and good signal-to-noise ratios.
The homodyne method, which consists in modulating the excitation light
and the detector at the same frequency, is routinely used in many biological
and biophysical laboratories because it can be performed with wide-field de-
tectors (such as a modulated intensified CCD camera). This approach has been
implemented in classical wide-field fluorescence microscopy, which enables
rapid FLIM image acquisition. 57-60 However, one limitation inherent in
this system is the nonconfocality of the excitation and, consequently, of the
fluorescence emission. To improve the axial discrimination of the FD
FLIM system, it can be simply combined with either a single-plane
illumination strategy 61 or a spinning disk module 62,63

(an example of such

an implementation is presented in Fig. 5.15 ).
Nowadays, several companies (ISS, Intelligent Imaging Innovations,
Lambert Instruments) have introduced commercial FD FLIM systems that
can be fully integrated with all commercial multiphoton, confocal, or
wide-field microscopes.

5. DATA ANALYSIS
5.1. Ratiometric imaging
Data processing methods for reaching quantitative FRET measurements be-
tween two fluorescent molecules with ratiometric imaging have been
exhaustively covered. 64-66 Briefly, in order to correct for both varying,
unknown concentrations of the donor and acceptor and instrumental
artifacts (e.g., spectral bleed-through), a large number of methods have
been proposed with different numbers of images and thus different levels
of complexity. 64,65,67 Among these, the most robust and widely used
method requires the acquisition of three fluorescence images. In this case,
the ratio R is defined by 68