20040400 XMUGXQ PFS0601 Principles of Fluorescence Spectroscopy Chemistry Department XMU.
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Transcript of 20040400 XMUGXQ PFS0601 Principles of Fluorescence Spectroscopy Chemistry Department XMU.
20040400
XMUGXQ PFS0601
Chapter Seven
Measurement of Fluorescence Lifetime
&Time-domain Fluorescence
&Frequency-domain
Fluorescence
20040400
XMUGXQ PFS0601
7.1 introduction
7.2 pulse lifetime measurement
7.3 Phase and Modulation Measurements of Fluorescence Lifetime
7.4 Measurement of Time-resolved Decays of Fluorescence
7.5 Application of Time-resolved Fluorescence
7.6 Phase-sensitive Detection of Fluorescence
7.7 Application of PSDF
Content
20040400
XMUGXQ PFS0601
7.1 Introduction
Information given by fluorescence lifetime
The frequency of collisional encounters
The rate of energy transfer
The rate of excited state reaction
Information related to its environment
And so on
20040400
XMUGXQ PFS0601
Methods for measuring fluorescence lifetime
Pulse method
Harmonic or phase-modulation method
I0
t
I0
t
Light source
Light source
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XMUGXQ PFS0601
7.2 pulse lifetime measurement
0
20
40
60
80
100
120
- 5 15 35 55
t/ ns
N
)()()(
tNkΓdt
tdNnr
/0 )()( tetNtN
S0
S1
S1
hvA hvF knr
Log F(t) or log N(t)
t
)/1(
)(
or
kΓ nr
20040400
XMUGXQ PFS0601
Average lifetime
ii
iii
tN
tNtt
)(
)(
ns
t
23
131211
1 ns, 1
2 ns, 1
3 ns, 1
For a large number of fluorophores and small time interval, this sum becomes
0
/
0
/
0
0
)(
)(
dte
dtte
dttN
dtttNt
t
t
20040400
XMUGXQ PFS0601
Multi-component
it
iietF /)(
t / ns
N
Pre-exponential factor
t / ns
ln F
(t)
5 ns
50 ns
iii
iii
t
2
20040400
XMUGXQ PFS0601
7.3 Phase and modulation measurements of fluorescence lifetime
Phase angle () Demodulation factor (m)
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XMUGXQ PFS0601
Phase-modulation method
p tan tan1p2/122 ]1[ mm 2/121 ]1)/1[( mm
=2f an important factor
For small lifetime, set large modulation frequency
For large lifetime, set small modulation frequency
Choosing modulation frequency, let
m = 0.3 ~ 0.7, = 30 ~ 70º
For commercial frequency-domain instrument, changing , measuring mi and i, calculate average lifetime
20040400
XMUGXQ PFS0601
7.4 Measurement of Time-resolved Decays of Fluorescence
Pulse lifetime measurement
Pulse width
I0
t
ps, fsEnough shorter compare to decay of fluorescence
Photon counts
Enough for accurate measurement Repeat excite
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XMUGXQ PFS0601
Pulse sampling Method
Photomultiplier Photomultiplier
Multichannel analyzer MACMultichannel analyzer MAC
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XMUGXQ PFS0601
Single photon counting method
Time to amplitude Time to amplitude converter TACconverter TAC
Multichannel pulse heiMultichannel pulse heigh analyzer MCPHAgh analyzer MCPHA
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XMUGXQ PFS0601
Streak Camera
Simultaneous measurements of both wavelength and time resolved decays
20040400
XMUGXQ PFS0601
Analysis of time-resolved decays of fluorescence intensity
0
20
40
60
80
100
120
- 5 15 35 55
t/ ns
N
In principle, for single exponential decay
tNe
N ,1
0
Log F(t) or log N(t)
t
)/1(
)(
or
kΓ nr
20040400
XMUGXQ PFS0601
Analysis of time-resolved decays of fluorescence intensity
t / ns
NIntensity profile of light, L(t)
Intensity decay of fluorescence, F(t)
Measured intensity decay, R(t)
In practice, in consideration of the pulse width of lamp and multi-exponential decay
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XMUGXQ PFS0601
Analysis of time-resolved decays of fluorescence intensity
Analysis of time-resolved decays of fluorescence intensity
tdttFtLtRt
)()()(0
In practicing measurement
Measuring the lamp profile, L(t), by using a solution which scatters light
Measuring the total intensity decay, R(t), by using the sample
)()()()( iii tttttFtLtR
At ti, a large number of pulses with equal width ti, each induce an impulse response in the sample
t - ti, emission delay compare to excitation
20040400
XMUGXQ PFS0601
Analysis of time-resolved decays of fluorescence intensity
Analysis of time-resolved decays of fluorescence intensity
tttFtLtR ii
tt
t
i
)()()(0
Total intensity decay,
t
dFtLtR
ttt
0
)()()(
,0
Commercial software available
The purpose is to get F(t)
20040400
XMUGXQ PFS0601
Analysis of time-resolved decays of fluorescence intensity
Analysis of time-resolved decays of fluorescence intensity
itn
iietF /
1
)(
Least –squares analysis of time-resolved decays
Let the number of components to be n,
Give initial values to i and i, and calculate, get
t
c dFtLtR0
)()()(
L(t), was measured
20040400
XMUGXQ PFS0601
Analysis of time-resolved decays of fluorescence intensity
Analysis of time-resolved decays of fluorescence intensity
The i and i values are varied until the best fit is observed.
n
i i
ici
n
iici
i
tR
tRtR
tRtR
1
2
1
22
2
)(
)]()([
)]()([1
A minimum value of 2 indicates the best fit.
In this expression the sum extends over the number (n) of channels or data points used for a particular analysis.
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XMUGXQ PFS0601
)(
)()(
tR
tRtRc
Analysis of time-resolved decays of fluorescence intensity
Analysis of time-resolved decays of fluorescence intensity
20040400
XMUGXQ PFS0601
7.5 Application of time-resolved fluorescence
Deduct back ground
I
t
LightLight
Back groundBack ground
Target componentTarget component
Gated time
Sampling time
Measure intensity ex/em
Boxcar integrator
Fast scan ex/ scan em
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XMUGXQ PFS0601
Example
荧光素标记荧光免疫分析,常常受到血液样品中胆红素背景荧光干扰,采用时间分辨荧光免疫分析可以有效地消除干扰。
荧光素寿命 3.6±0.46 ns, 测定时间 6.0 ns
胆红素寿命 0.21±0.14 ns, 测定时间 信号为零
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XMUGXQ PFS0601
Application Dynamic quenching
][1 QKSV
0
25
50
75
100
0 20 40 60
t/ ns
N
[Q]
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XMUGXQ PFS0601
Application
Time-resolved fluorescence immunoassay
TBP-Eu3+
XL665
链 [ 霉 ] 亲和素
Example
TBP-Eu3+
D A
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XMUGXQ PFS0601
Example
Interference:
Background from media
Emission from free acceptor
20040400
XMUGXQ PFS0601
7.6 Phase-sensitive detection of fluorescence
Principle
F(t)
I(t)
F(t)
mL=b/a
mA=B/A
mB=B’/A’
mL>mA>mB
A<B
A<B
20040400
XMUGXQ PFS0601
principle
tbatI sin)(
)sin(1)( tmmtF L
)cos()( DD iikFF
For single component
Excited with
Emission
Phase sensitive detection (lock-in amplifer )
0)cos(90
1)cos(,0
DD
DD D, detection phase
20040400
XMUGXQ PFS0601
Principle
0)cos(90
1)cos(,0
DD
DD
)cos()( DD iikFF
At i
F
F change with cos(D-)
At i
F change with
F
20040400
XMUGXQ PFS0601
principle
)sin()()sin()(),( BBBAAA tmFtmFtF
)cos()()cos()(),( BDBBADAAD mFmFF
90)( AD
For two component
Emission
Phase sensitive detection
Phase depression
)90cos()(),( BABBD mFF