Biology Reference
In-Depth Information
with both methods (heterodyne and homodyne method), calibration of the
FLIM system with a reference of known fluorescence lifetime t
ref
is indis-
pensable for taking into account the phase shift and modulation introduced
by the electronics and optics (cf. Eqs.
5.31 and 5.32
). The fluorescence
lifetime of an unknown sample is then defined by
77
r
m
r
ef
m
2
1
o
2
2
ref
t
m
¼
1
þ
o
t
1
½
5
:
33
1
o
tan
1
t
'
¼
tan
''
ref
þ
ð
ot
ref
Þ
½
5
:
34
where
'
ref
and
m
ref
are, respectively, the phase and modulation values esti-
mated from the reference. The fact that these fluorescence lifetimes (t
m
and
t
'
) are calculated for each pixel must be emphasized. The resulting FLIM
image, which is displayed using a color scale, is usually superimposed on
the intensity image in order to highlight the brightest regions (see
Fig. 5.17
).
When using the FD FLIM system to record biosensor activity in living
cells, evolutions in time of the phase and modulation lifetimes (t
'
and t
m
) are
sometimes represented.
73
However, this method gives just an indication of
the relative changes between the two states of the biosensor. In fact, these
phase and modulation lifetimes acquired during a single-frequency experi-
ment do not correspond to the true lifetimes of the biosensor. To recover
these true lifetime components, it is necessary to acquire multiple frequency
FLIM images and to fit pixel by pixel the experimental phase
and mod-
ulation
m
for each frequency owith a function of o
.
59
Experimental data are
usually adjusted to the theoretical values by minimizing an error function
(using a Levenberg-Marquardt algorithm) without any
a priori
information
on the lifetime components.
59,78,79
If it can be assumed that the lifetime
information is the same for all pixels of the FLIM image, data can be
globally analyzed.
80,81
In both cases, obtaining reliable lifetime
components is time consuming and it is hardly accessible to the nonexpert.
'
5.2.2 Time-domain lifetime imaging
5.2.2.1 Extracting lifetime components
In time-domain methods, fluorescent samples are repeatedly excited by
short pulses of light, and the resulting fluorescence intensity decay histo-
grams can be recorded for each pixel of the FLIM image with different de-
tectors (see
Section 4
). Regardless of the technique employed, experimental
lifetime components are usually deduced by adjusting the experimental
decay histograms with the theory. In TD FLIM, the theoretical detected
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