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d
and
A
d
, respectively;
Fig. 8.5B
).
104,105
From the difference in phases, a fluorescence lifetime is given by
phases and ratio in amplitudes are
y
tan
y
d
o
t
;y
¼
;
½
8
:
21
d
q
1
=A
d
2
ð
Þ
1
t
d
;
A
¼
:
½
8
:
22
o
where
d,
A
represent phase and modulation lifetimes, which are a
unique value in the case of fluorescent proteins with a single lifetime com-
ponent (
Fig. 8.5B
). One of the problems inherent in the frequency-domain
method is that the excited state of CFP derived from
Aequorea
GFP decays
with multiple (three or four) time constants, making frequency-domain life-
time measurements of FRET using CFP as a donor complicated. Several
fluorescent proteins that can be substituted for CFP, including Cerulean
and mTurquoise, are reported to possess a single lifetime component.
106,107
t
and
t
d,
y
6.4. Lifetime measurement using a flow cytometer
Although fluorescence microscopy has been and continues to be the main
tool used to perform FRET experiments, the number of cells that can be
observed within a period is restricted. For quantitative measurement, tech-
niques aimed at obtaining FRET indices with high throughput may be nec-
essary. Fluorescence flow cytometry is a good candidate for handling a vast
number of cells in a short period; however, subtle studies regarding the suc-
cessful application of this technique are available. We would therefore like to
introduce a unique instrument, the world's first flow cytometer designed to
detect fluorescence lifetime along with fluorescence intensity, which we are
now developing. This machine, named “Flicyme,” for fluorescence lifetime
cytometer (
Fig. 8.5C
), possesses great potential for the highly quantitative
observation of FRET efficiency. In addition, it can be easily utilized for
high-throughput drug screening, especially for molecular targeted drugs.
Flicyme determines the fluorescence lifetime and fluorescence intensity
for each individual living cell that passes through a flow cell at high speed.
Key specifications of Flicyme are listed in
Table 8.2
. Cells are illuminated by
the diode laser (407 or 440 nm, 60 mW) with a modulation frequency of
28 MHz, which results in the acquisition of lifetime with a precision of
0.02 ns and at a maximum rate of 10,000 cells/s. These achievements are
highly advantageous for facilitating FRET experiments in many situations,
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