Biology Reference
In-Depth Information
A1 (mono,
n
= 0)
t
mean
= 1.81 ns
á
t
ñ
= 1.81 ns
t
=0
t
=90
2.5
100
8
a
1
t
mean
t
mean
<
t
>
10
6
2.0
1
1
4
0
t
1
t
=0
t
=90
2
=1.60
c
-
3
1.5
1000
2
t
A2 (mono,
n
= 2)
t
mean
= 1.71 ns
á
t
ñ
<
t
>
2
= 1.71 ns
100
0
0
0.5
1
1.5
2
2.5
3
3.5
1.0
10
Lifetime (ns)
D
2.1
1
B
3
2.0
0
2
=1.55
c
-
3
1.9
1000
A3 (bi,
n
= 2)
t
mean
= 1.49 ns
á
t
ñ
= 1.79 ns
1.8
100
1.7
10
1.6
t
mean
(ns)
1
3
1.5
<
t
>
(ns)
20
m
m
0
2
=1.08
c
-
3
1.4
0
20
40
60
80
0
2
4
6
8
10
t
(min)
t
(ns)
Figure 5.18 Data analysis of fluorescence lifetime measurements acquired with the time-correlated single-photon counting (TCSPC) method.
(A1) Typical experimental intensity decay (dots) acquired in a fewmilliseconds per pixel (corresponding to an acquisition time of 300 s for the
complete image represented in (B); the fit obtained with standard least squares method (LSM) is indicated with a line. Because of the small
photon count
N
, the error function (
w
2
) is nonflat and elevated, indicating that the adjustment is not perfect. (A2) One possibility to increase
N
consists in applying a spatial binning of factor
n
(sum all pixels comprised in a (2
n
þ1)
2
squared region). In this case, the fit is slightly improved
but
w
2
is still nonflat because the monoexponential model is not adapted. (A3) When the biexponential model is used, the fit is satisfactory,
which means that the sample proportion and lifetime can be correctly estimated. (B) Fluorescence intensity image of living cells transfected
with
T
Epac
VV
. The distributions of all fitting parameters (
a
1
,
t
1
, and
t
2
) for the complete image are shown in (C). Also represented are the mean
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