20040200

XMUPFS01-ITF02

Principles of Fluorescence Spectroscopy

Chemistry Department

XMU

20040200

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

20040200

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

20040200

XMUPFS01-ITF02Absorption and emission spectra

20040200

XMUPFS01-ITF02Excitation and emission spectra

1 2

34

Excitation spectrum Emission spectra

F

F

em

20040200

XMUPFS01-ITF02Three dimension spectrum

20040200

XMUPFS01-ITF02Three dimension spectra

引自林竹光等人的论文

ex

/ n

m

em / nm

20040200

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

20040200

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.

20040200

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

20040200

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

20040200

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-

20040200

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

20040200

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

20040200

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

20040200

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

20040200

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

20040200

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

20040200

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

20040200

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

20040200

XMUPFS01-ITF02Magic angle 魔鬼角

2

1cos3 2

r

5

3cos2

0,7.54

1,0

r

r

20040200

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

20040200

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

20040200

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 ……

20040200

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

20040200

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

20040200

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

20040200

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

20040200

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).

20040200

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

20040200

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

20040200

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).

20040200

XMUPFS01-ITF021.9 Intensity and concentration

shiftstokesPrΦemex ',,,,,,

Principle of quantitative and qualitative analysis by fluorometry

Qualitative probing parameters:

Quantitative determination

bcΦIF 303.20

20040200

XMUPFS01-ITF021.10 Intrinsic fluorophore and extrinsic fluoroph

oreIntrinsic fluorophores are those which occur naturally

Extrinsic fluorophores, fluorescence probes

20040200

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

20040200

XMUPFS01-ITF02Protein fluorescence

20040200

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

20040200

XMUPFS01-ITF02membrane

20040200

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.

20040200

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

20040200

XMUPFS01-ITF02Other fluorophores

Fluorescein

OHO O

CO2H

(Z) (Z)

(E)

N(CH3)2

SO2Cl

Dansyl chloride 丹磺酰氯

Chlorophyll 叶绿素

20040200

XMUPFS01-ITF02Fluorescent indicators

Fluorophores whose spectral properties are sensitive to a substance of interest, such as K+, Ca2+, Mg2+, Na+, Cl-, O2 and CO2

…….