Principles of Fluorescence Spectroscopy
44
20040400 XMUGXQ PFS0601 Principles of Fluorescence Spectroscopy Chemistry Department XMU
description
Principles of Fluorescence Spectroscopy. Chemistry Department XMU. Chapter Seven. Measurement of Fluorescence Lifetime & Time-domain Fluorescence & Frequency-domain Fluorescence. Content. 7.1 introduction 7.2 pulse lifetime measurement - PowerPoint PPT Presentation
Transcript of Principles of Fluorescence Spectroscopy
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Principles of Fluorescence Spectroscopy
Chemistry Department
XMU
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Chapter Seven
Measurement of Fluorescence Lifetime
&Time-domain Fluorescence
&Frequency-domain
Fluorescence
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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
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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
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Methods for measuring fluorescence lifetime
Pulse method
Harmonic or phase-modulation method
I0
t
I0
t
Light source
Light source
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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
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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
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Multi-component
it
iietF /)(
t / ns
N
Pre-exponential factor
t / ns
ln F
(t)
5 ns
50 ns
iii
iii
t
2
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7.3 Phase and modulation measurements of fluorescence lifetime
Phase angle () Demodulation factor (m)
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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
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Phase-modulation method
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Phase-modulation method
Single component
mp
multicomponent
Average lifetime
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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
XQGuo
两次激发之间的时间间隔至少大于荧光衰变的5倍。每次激发后，光电倍增管通过延迟线路控制，在一定时间里打开，记录信号，并将其储存在多到分析器中；再次激发，光电倍增管在另一个不同的时间里打开，记录信号，然后存在多道分析器中。通过反复的激发，在不同的时间里打开光电倍增管记录信号，这样就可以记录下荧光体的衰变动力学曲线。
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Pulse sampling Method
Photomultiplier Photomultiplier
Multichannel analyzer MACMultichannel analyzer MAC
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Single photon counting method
Time to amplitude Time to amplitude converter TACconverter TAC
Multichannel pulse heiMultichannel pulse heigh analyzer MCPHAgh analyzer MCPHA
XQGuo
单光子计数的方法，仍然采用脉冲技法，测量光子到达光电倍增管的时间。将强度调节至只有一个光子作用于光电倍增管，并记录从激发至光子到达光电倍增管的时间。反复激发，每次记录一个光子的到达时间。相当于激发一个荧光体群体，每个光子在不同的时间到达阴极，这样就相当于记录下这个荧光群体的动力学衰变曲线。假设激发一个荧光体，每次受激后在激发态停留的时间是随机的，有的时间长，有的时间短，但无数次激发的衰变满足单指数衰变。
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Streak Camera
Simultaneous measurements of both wavelength and time resolved decays
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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
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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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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
XQGuo
t-t',发光时间相对于激发延迟
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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)
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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
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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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)(
)()(
tR
tRtRc
Analysis of time-resolved decays of fluorescence intensity
Analysis of time-resolved decays of fluorescence intensity
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Example Example
Single exponential decay
Double exponential decay
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ExampleExample
Single exponential decay
Double exponential decay
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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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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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Application
Multi-components measurement
I
t
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Application Dynamic quenching
][1 QKSV
0
25
50
75
100
0 20 40 60
t/ ns
N
[Q]
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Application
Time-resolved fluorescence immunoassay
TBP-Eu3+
XL665
链 [ 霉 ] 亲和素
Example
TBP-Eu3+
D A
XQGuo
GST HDM2研究GST融合HDM2蛋白与P53蛋白质之间的作用， 采用荧光共振能量转移模式。受体背景荧光干扰，借助于受体能量转移荧光的长寿命特征与游离的受体荧光区别。铀化合物通过抗GST抗体与GST特异作用标记到GST融合HDM2蛋白上。XL665(别藻蓝蛋白连接化合物）通过链[霉]亲和素标记到P53蛋白上。通过能量转移的效率研究两种蛋白之间的作用。
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Example
Interference:
Background from media
Emission from free acceptor
XQGuo
背景消除：时间分辨，消除游离受体的荧光比率测定，消除介质诱导的能量转移
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Application background
Free XL665
TBP-Eu3+(665nm/620nm)
FRET(665nm/620nm)
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Expression for the Figs
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Reference
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Time-resolved emission spectra
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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
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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
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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
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principle
)sin()()sin()(),( BBBAAA tmFtmFtF
)cos()()cos()(),( BDBBADAAD mFmFF
90)( AD
For two component
Emission
Phase sensitive detection
Phase depression
)90cos()(),( BABBD mFF
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Phase sensitive detection fluorescence spectra
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Example
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Example
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Example
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Example
