Biology Reference
In-Depth Information
6.2. Time domain
Changes in the fluorescence intensity (or the number of photons emitted) of
the donor chromophore [
F
d
(
t
) (photons cm
-3
s
-1
)] excited by the ideal pulsed
light source at time point 0 can be given by
or simply
F
d
t
F
0
exp
t
t
d
exp
t
t
d
F
d
t
ðÞ¼
ðÞ/
;
½
8
:
18
where
F
0
(photons cm
-3
s
-1
) represents the fluorescence intensity at time
point 0 (s) (i.e., the product of the number of excited electrons of fluores-
cence molecules (electrons cm
-3
) and the rate constant of the radiative
transition state (s
-1
)).
103
Thus, the observed fluorescence decays exponen-
tially (
Fig. 8.4A
). Also, the change in fluorescence intensity of the donor
in the presence of acceptor
F
d
0
t
ðÞ
is given by
or
F
d
0
t
:
t
t
d
0
t
t
d
0
F
d
0
t
ðÞ¼
F
0
exp
ðÞ/
½
:
exp
8
19
When the proportion of the donor-bound acceptor is given by
a
(un-
bound form, 1 -
a
), the time-dependent change in donor fluorescence in-
tensity is therefore given by
þ
:
t
t
d
0
t
t
d
F
d
t
ðÞ/
a
exp
ð
1
a
Þ
exp
½
8
:
20
3 [ns]) and
F
d
(
t
)
0
(
t
d
0
¼
1 [ns]), as well as
F
(
t
) when
a
is 0.3, are shown in
Fig. 8.3A
. Thus, donor occupancy (
a
) can be calculated
by fitting the fluorescence decay obtained by fluorescence lifetime imaging
microscopy (FLIM). The FLIM method of time domain is often combined
with two-photon excitation microscopy, which is
Examples of
F
d
(
t
)(
t
d
¼
coupled with
femtosecond-pulsed lasers.
6.3. Frequency domain
In contrast, in the frequency-domain method, time-dependent changes in
fluorescence intensities in response to modulated excitation power are con-
tinuously acquired. Therefore, this method can be used with a wide-field
fluorescence microscope equipped with faster image acquisition devices
such as an image intensifier or electron multiplying CCD (EMCCD). When
fluorescent proteins (or dyes) are excited by a sine wave with an angular fre-
quency of
(s
-1
), the fluorescence signal output is elicited as a sine curve
with the same frequency but a distinct phase and amplitude (difference in
o
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