Biology Reference
In-Depth Information
6.1. Donor fluorescence lifetime as a measure of FRET
efficiency
Measurement of the lifetime of donor fluorescence is the most reliable
method to “quantify” FRET efficiency. 102 Lifetime measurement is inde-
pendent of chromophore concentrations and thus quantitatively comparable
from sample to sample. In this approach, E is given by
d 0
t
t
E
¼
1
d ;
½
8
:
17
t d 0 and
where
t d are the donor lifetimes in the presence and absence of the
acceptor, respectively. After excitation by a short laser pulse, the excited-
state donor (when it contains a single component of lifetime) decays with
a single exponential time course (
t
d ). FRET results in a short lifetime
t d 0 ) (see below and Fig. 8.4A ).
(
A
B
w
F d = exp (
/ 3)
F d ¢ = exp ( - t / 1)
- t
A d =
a
/
A
q d
F t0 exp [ - ( t - t 0 )/ t ]
Excitation
A
1/
e
a
0246810
(ns)
C
Stained cells
ADC & PC
Sheath fluid
F d = 0.7 exp ( - t / 3)
F d ¢ = 0.3
PMT
PMT
exp (
- t
/ 1)
Excitation
laser oscillator
Demodulation
signal
processing
board
F d + F d ¢
Excitation
Intensity-
modulated
laser
Forward scatter signal
Intensity-modulated
fluorescence
Laminar
sheath flow
in cuvette
Photo diode
02468 0
(ns)
Figure 8.4 FRET evaluation by fluorescence lifetime. (A) Upper panel: time-dependent
decay of fluorescence intensity with lifetimes of 3 ns (black line) and 1 ns (gray) in re-
sponse to pulsed excitation (dashed). Lower panel: the resulting averaged fluorescence
lifetime (solid line) when the bound fraction (with a fluorescent lifetime of 1 ns) and
unbound fraction (3 ns) are mixed in the ratio 3:7. (B) Principle of the frequency-domain
method for the measurement of fluorescence lifetime. Modulated fluorescent emissions
(black), in response to modulated excitations (gray) with a circular frequency of
, are
shown, along with phase differences and amplitude ratios. (C) A cytometer for lifetime
measurement, Flicyme. Exterior view (left) and interior optical paths with electronic
circuits (right) are shown.
o
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