principles of fluorescence spectroscopy
DESCRIPTION
Principles of Fluorescence Spectroscopy. Chemistry Department XMU. Introduction to Fluorescence. 1.0 Introduction 1.1 Phenomenon of Fluorescence 1.2 Excitation and Deactivation of molecule 1.3 Models of Molecular emission 1.4 Characteristics of Fluorescence Emission - PowerPoint PPT PresentationTRANSCRIPT
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XMUPFS01-ITF02
Principles of Fluorescence Spectroscopy
Chemistry Department
XMU
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XMUPFS01-ITF02Introduction to
Fluorescence• 1.0 Introduction• 1.1 Phenomenon of Fluorescence• 1.2 Excitation and Deactivation of molecule• 1.3 Models of Molecular emission• 1.4 Characteristics of Fluorescence Emission• 1.5 Characteristics of Fluorophore• 1.6 Fluorescence quenching• 1.7 Resonance energy transfer• 1.8 Time scale of Fluorescence• 1.9 Intensity and Concentration• 1.10 Fluorophore
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XMUPFS01-ITF021.5 Characteristics of fluorophore
1.5.1 Excitation wavelength 激发波长 ex
1.5.2 Emission wavelength 发射波长 em
1.5.3 Extinction (absorption) coefficient 吸光系数 1.5.4 Stokes’ shift
1.5.5 Fluorescence lifetime 荧光寿命
1.5.6 Fluorescence quantum yield 荧光量子产率
1.5.7 Fluorescence Anisotropy 各项异性 r
15.8 Fluorescence Polarization 荧光偏振 p
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XMUPFS01-ITF02Absorption and emission spectra
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XMUPFS01-ITF02Excitation and emission spectra
1 2
34
Excitation spectrum Emission spectra
F
F
em
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XMUPFS01-ITF02Three dimension spectrum
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XMUPFS01-ITF02Three dimension spectra
引自林竹光等人的论文
ex
/ n
m
em / nm
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XMUPFS01-ITF021.5.5 Fluorescence lifetime
0
20
40
60
80
100
120
- 5 15 35 55
t/ ns
N
/0 )()( tetNtN
Definition
Lifetime for Single molecule: the time the molecule spends in the excited state prior to return to the ground state.
Average lifetime: the average time the molecule spends in the excited state prior to return to the ground state.
Average fluorescence lifetime
etNNt
1)()(, 0
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XMUPFS01-ITF02Expression
nrkΓ
1
relaxation (10-12 s)
S0
S1
S1
hvA hvF knr
Simplified Jablonski diagram
: Emission rate
Knr: nonradiative decay rate
Lifetime Intrinsic or natural lifetime
Γ
10
The lifetime of the fluorophore in the absence of nonradiative processes is called the intrinsic lifetime.
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XMUPFS01-ITF02
1.5.6 fluorescence quantum yield
nrkΓ
ΓΦ
Definition
The fluorescence quantum yield is the ratio of the number of photons emitted to the number absorbed
Expression
Relationship with lifetime
nrkΓ
1Γ
10
0
Φ
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XMUPFS01-ITF02Determination Comparison with a standard
1. Chose a Standard with a quantum yield s
2. At I determine the absorption (As) of the standard
3. At ex = I excited the standard, and integrate the emission spectrum of standard, get F
4. Repeat 3th step with the blank solvent. Minus the emission from blank, get Fs
F
5. Repeat 2-4th steps with the sample, and get Ax and Fx
s
x
x
Ssx F
F
A
AΦΦ
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XMUPFS01-ITF02Choose Standards
Enough absorbance both standard and sample at chosen excited wavelength.
Moderate quantum yield.
1. 0.05 mol / L sulfate of quinine, = 0.55
Examples
2. RuPy3Cl2, deoxygenated solution, 20C, = 0.042
N
N
NN
N
NRu2+
2Cl-
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XMUPFS01-ITF021.5.7 Fluorescence Anisotropy
and Fluorescence Polarization
光是一种电磁波,具有相位相同的两个互相垂直的振动矢量,电矢量和磁矢量
光的性质
EH
偏振光 非偏振光
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XMUPFS01-ITF02荧光分子
荧光分子可以 看成是一个振荡偶极子( oscillating dipole)
吸收偶极距 absorption dipole moment
发射偶极距 emission dipole moment
基态与激发态的电子分布不同,分子的激发跃迁距和发射跃迁距往往是不共线的
CH
CH
CH
CH
CH
CH 吸收跃迁距和发射跃迁距共线
AE
当不存在旋转运动时,吸收跃迁距与发射跃迁距之间的夹角对每一个荧光分子而言是固定的。
吸收跃迁距 absorption transition moment
发射跃迁距 emission transition moment
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XMUPFS01-ITF02Principle of photoselective excitation
Fluorophores preferentially absorb photons whose electric vectors are aligned parallel to the transition moment of the fluorophore.
EM, Eba M
光吸收选择示意a. 吸收几率∝ M , b. 吸收几率∝ Mcos2
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XMUPFS01-ITF02
z
激发偏振器
发射偏振器
x
y
检测器
I
I
Definition
0.1,0 PrI
0,11 PrII
II
IIP
II
II
II
IIr
2II
II
polarization
anisotropy
5.0,4.0 Pr
For fluorophore
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XMUPFS01-ITF02Polarization and anisotropy
r
rP
2
3
P
Pr
3
2
荧光偏振与荧光各向异性可通过以下公式相互转换:
当体系中存在多种荧光体时,所测得的荧光各向异性是各种荧光体荧光各向异性的平均值:
i
iirfr
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XMUPFS01-ITF02各项异性的物理意义
所测得的各项异性,反映两种取向:吸收跃迁距相对于光子电矢量的取向。
对分子随机取向的溶液体系而言,无特性。
CH
CH
CH
CH
CH
CH
分子内固有吸收跃迁距和发射跃迁距共线, 若以偏振光激发, 且分子不发射旋转运动,应有 r = 1, 但实际上, r = 0.4 , 原因, 分子随机取向。
DPH
问题的说明
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XMUPFS01-ITF02Isotropic solution
以平行于 z 轴的偏振光激发荧光体时,激发态荧光体布居是围绕 z 轴呈对称分布的 激发分子的分布可用下式表示,
f ( )d = cos2 sin d
荧光体的几率分布
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XMUPFS01-ITF02坐标系中的荧光体
I (,) = cos2
I (,) = sin2 sin2
以 z 轴对称分布
2
1sin 2
0
2
0
2sin2
d
d
2
2/
0
2
cos
cos)(
dfI II
2
2/
0
2
sin2
1
sin)(2
1
dfI
2
1cos3 2
r
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XMUPFS01-ITF02Magic angle 魔鬼角
2
1cos3 2
r
5
3cos2
0,7.54
1,0
r
r
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XMUPFS01-ITF02激发跃迁距相对于发射跃迁距取向
固有取向(),分子内在性质
r = 0.4
A,E
A
E r < 0.4
0
22
)2
1cos3)(
2
1cos3( rr
旋转运动(),环境性质
= 0 > 0
)2
1cos3(
2
0
rr
A E
E
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XMUPFS01-ITF02Losing of anisotropy
In fluid solution, most fluorophores roatate extensively in 50 – 100 ps.
What happens to anisotropy?What happens to anisotropy?
If a fluorophore is bound to a macromolecule, such as human serum albumin, whose rotation correlation time() is 50 ns, what happens to the anisotropy?
)/(10
rr
Perrin equation
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XMUPFS01-ITF021.6 Fluorescence quenching
quench
Decreases in fluorescence intensity are called quenching
Quencher
Stern-Volmer equation ][10 QKF
FST
relaxation (10-12 s)
S0
S1
S1
hvA hvF knr
Q
Q
kq[Q]
Other molecules colliding or reaction with fluorophores, causing quench Oxygen, halogens ……
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XMUPFS01-ITF021.7 Fluorescence resonance energy
transfer
)()()(
)()( 100
10 SASDSA
SDSD
Energy transfer
Conditions for energy transfer
Distance 60 )(1
r
RK
DT
R0 Forster distance
r distance, A and D
Overlap of spectra, A’s absorption and D’s emission D’s emission A’s absorption
D, Donor; A, Acceptor
Orientation
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XMUPFS01-ITF021.7 Fluorescence resonance energy
transfer
relaxation (10-12 s)
S0
S1
S1
hvA hvF knr
A(S0)
A(S1
)
60 )(1
r
RK
DT
Process of deactivation
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XMUPFS01-ITF021.8 Time scale of molecular process in
solution e.g. Collisional quenching of fluorescence by oxygen
Diffusion coefficient (D) of O2 at 25C is 2.5 x 10-5 cm2/s. the average distance (x2) an O2 can diffuse in 10 ns given by the Einstein equation,
7022 Dx Å
Which is comparable to the thickness a biological membrane or the diameter of a protein.
e.g. Fluorophore-solvent interaction during the lifetime of fluorophores probing micro-envirnoment, pH, viscosity, polarity, et al.
Provide a way probing resolved oxygen
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XMUPFS01-ITF02Probing pH
马丽华,温珍昌,孙向英,江云宝,高等学校化学学报,2001 , 22 ( 7 ), 1125-1127
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XMUPFS01-ITF02Probing polarity
550 600 650 700 750 8000.0
0.2
0.4
0.6
0.8
1.0
CH3(CH
2)
3OH
CH3(CH
2)
5OH
CH3(CH
2)
9OH
DPPGDPPG / Chol.
In buffer
[Ru(bpy)2(dppz)]2+ in
Solvents or Vesicles,
EX=463 nm, 20 oC
Em
issi
on In
tens
ity
Emission Wavelength (nm)
●Xiang-Qun Guo, F. N. Catellana, L. Li and J. R. Lakowicz, A long-lifetime Ru(II) metal-ligand complex as a membrane probe , Biophysical Chem., 71, 51-62, (1998.3).
●Xiang-Qun Guo, F. N. Catellana, L. Li and J. R. Lakowicz, A long-lifetime Ru(II) metal-ligand complex as a membrane probe , Biophysical Chem., 71, 51-62, (1998.3).
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XMUPFS01-ITF021.8 Time scale of molecular process in
solution
F
Ag F-Ag
Ab
F-Ag:Ab
发射消偏振旋转快
旋转慢
保持偏振发射
垂直偏振激发
e.g. Rotation diffusion of protein during the lifetime of fluorophores
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XMUPFS01-ITF021.8 Time scale of molecular process in
solution
0 10 20 30 40 50 600
2
4
6
41 oC
RuDPPG vesicle containingcholesterolEX=463nm, EM=650nm,
0% 20% 33% 50%
Em
issi
on I
nten
sity
T oC
e.g. Rotation diffusion of fluorophore in the excited-state lifetime, probing properties of membrane.
Emission intensities of DPPG vesicles labeled with of [Ru(bpy)2(dppz)]2+ at various cholesterol concentrations, measured as a function of increasing temperature towards the lipid phase transition temperature
Reference
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XMUPFS01-ITF02Structure of DPPG
N N
H3C CH3
Re
COOC
NOC COOH
+
●Xiang-Qun Guo, F. N. Catellana, L. Li and J. R. Lakowicz, A long-lifetime Ru(II) metal-ligand complex as a membrane probe , Biophysical Chem., 71, 51-62, (1998.3).
●Xiang-Qun Guo, F. N. Catellana, L. Li and J. R. Lakowicz, A long-lifetime Ru(II) metal-ligand complex as a membrane probe , Biophysical Chem., 71, 51-62, (1998.3).
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XMUPFS01-ITF021.9 Intensity and concentration
shiftstokesPrΦemex ',,,,,,
Principle of quantitative and qualitative analysis by fluorometry
Qualitative probing parameters:
Quantitative determination
bcΦIF 303.20
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XMUPFS01-ITF021.10 Intrinsic fluorophore and extrinsic fluoroph
oreIntrinsic fluorophores are those which occur naturally
Extrinsic fluorophores, fluorescence probes
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XMUPFS01-ITF02Protein fluorescence
H2N CH C
CH2
OH
O
Indole group of tryptophan 色氨酸中的吲哚基团
H2N CH C
CH2
OH
O
HN
ex = 280, em = 340, highly sensitive to solvent polarity
H2N CH C
CH2
OH
O
OH
tyrosinePhenylalanine
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XMUPFS01-ITF02Protein fluorescence
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XMUPFS01-ITF02membrane
Typically do not display intrinsic fluorescence
Extrinsic membrane fluorescence probe
CH
CH
CH
CH
CH
CH
DPH
O(C2H5)2N N+(C2H5)2
CO2H
(Z) (Z)
(E)
Rhodamine B
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XMUPFS01-ITF02membrane
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XMUPFS01-ITF02DNA
NNH3C
H3CN
CH3
CH3
DNA is weakly fluorescent or nonfluorescent
Extrinsic fluorescence probe
Acridine orange
N+
H2N NH2
CH2CH3
Ethidium bromide
Staining of cells with dyes that bind to DNA is widely used to visualize and identify chromosomes.
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XMUPFS01-ITF02Cofactor 辅酶
Nicotinamide adenine dinucleotide 烟酰胺腺嘌呤二核苷酸
NADH, fluorescent; NAD+ nonfluorescent
Flavins 黄素( FAD, flavin adenine dinucleotide 黄素腺嘌呤二核苷酸 ; FMN, flavin mononucleotide 黄素单核苷酸 )
Related to enzymatic reaction, probing enzymatic process
As a intrinsic probe to study cell, tissue, and protein
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XMUPFS01-ITF02Other fluorophores
Fluorescein
OHO O
CO2H
(Z) (Z)
(E)
N(CH3)2
SO2Cl
Dansyl chloride 丹磺酰氯
Chlorophyll 叶绿素
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XMUPFS01-ITF02Fluorescent indicators
Fluorophores whose spectral properties are sensitive to a substance of interest, such as K+, Ca2+, Mg2+, Na+, Cl-, O2 and CO2
…….