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Principles of Fluorescence Spectroscopy. Chemistry Department XMU. Chapter Eight. Measurement of Fluorescence Anisotropy. Content. 8.1 Measurement of Anisotropy 8.2 Polarization Spectra of Fluorophores 8.3 Perrin Equation 8.4 Time-Dependent Decays of Anisotropy

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Presentation Transcript
chapter eight
XMUGXQ PFS0801Chapter Eight

Measurement of Fluorescence Anisotropy

content
XMUGXQ PFS0801Content

8.1 Measurement of Anisotropy

8.2 Polarization Spectra of Fluorophores

8.3 Perrin Equation

8.4 Time-Dependent Decays of Anisotropy

8.5 Nonsymmetric Fluorophores

8.6 Application of Anisotropy Measurement

8 1 measurement of anisotropy
XMUGXQ PFS08018.1 Measurement of Anisotropy
  • L-format or single-channel method

IVV = kSVI

IVH = kSHI

k是与荧光量子产率及仪器因素相关的比例常数。

measurement of anisotropy1
XMUGXQ PFS0801

DET

DET

Measurement of Anisotropy
  • T-format or two-channel method

Excited vertically

Gi, gain to i component

measurement of anisotropy2
XMUGXQ PFS0801

DET

DET

Measurement of Anisotropy

Excited horizontally

RH = G‖/G

RV/RH = I‖/I  

8 2 polarization spectra of fluorophores
XMUGXQ PFS0801

L

A

A

E

8.2 Polarization Spectra of Fluorophores
  • Anisotropy

For a dilute vitrified solution in which fluorophores are random oriented

polarization spectra of fluorophores
XMUGXQ PFS0801Polarization Spectra of Fluorophores

In vitrified solutions, the measured anisotropy values provide a measure of the angle between the absorption and emission dipoles.

The orientation of the absorption dipole differs for each absorption band and thus the angle varies with wavelength.

Anisotropy varies with the excitation wavelength.

polarization excitation spectrum
XMUGXQ PFS0801Polarization excitation spectrum

Polarization spectrum of perylene in propylene glycol ( 丙烯二醇)at -50ºC

polarization excitation spectrum1
XMUGXQ PFS0801Polarization excitation spectrum

Polarization spectrum of 9-vinyl anthracene in propylene glycol ( 丙烯二醇)at -50ºC

polarization emission spectrum
XMUGXQ PFS0801

hvex

hvem

A

F

Polarization Emission spectrum

Generally, the anisotropy is independent of the emission wavelength.

8 3 perrin equation
XMUGXQ PFS0801

A

E

E

8.3 Perrin equation

In the presence of rotational diffusion

On excitation, the time-resolved decay of anisotropy for a spherical molecule is a single exponential:

, rotational correlation time of the fluorophore

perrin equation
XMUGXQ PFS0801Perrin equation

For steady state measurement, the anisotropy is an average of r(t) by F(t).

Perrin Equation

perrin equation1
XMUGXQ PFS0801Perrin equation

For highly viscous solution or a macro-molecule,

Where  , / <<1

Otherwise,

When  <<,  / >>1

The observed anisotropy will be zero

perrin equation2
XMUGXQ PFS0801Perrin equation

The alternative forms of perrin equation

or

or

其中,R为荧光体的转动速率,-1 = 6R,为荧光体的旋转松弛时间,= 3。

nonsymmetric fluorophores
XMUGXQ PFS0801Nonsymmetric Fluorophores

Segmental mobility of biopolymer-bound fluorophore

其中,F和P分别为荧光分子作局部旋转运动和随大分子旋转运动的旋转相关时间。< 1 时,可以理解为荧光体的局部旋选运动受到一定的约束,荧光各项异性值衰减至一极值,r= r0 (1 - ),随后继续随大分子旋转,荧光各项异性值随时间衰减为零。= 1,荧光体的荧光各项异性值呈单指数衰减。

nonsymmetric fluorophores1
XMUGXQ PFS0801Nonsymmetric Fluorophores
  • Different fluorophores or fluorophore in different microenvironment

For a protein-bound fluorophore

r01 = r02

If lifetime not change on labeling

1 << 2

8 6 application of anisotropy measurement
XMUGXQ PFS08018.6 Application of Anisotropy Measurement
  • Estimation of the micro-viscosities of cell membranes
application
XMUGXQ PFS0801Application
  • Estimate the rotational diffusion and volume of protein
application1
XMUGXQ PFS0801

r

[P]

Application
  • Measurement of association reactions

K dissociation constant

rB

rF, in the absence of L

rB, at a high protein concentration, all L bind to protein

rF

application2
XMUGXQ PFS0801

r

r

[DNA]

T

Application
  • Denature of DNA

TC

application3
XMUGXQ PFS0801

F

Ab

Ag

F-Ag:Ab

F-Ag

发射消偏振

旋转快

垂直偏振激发

保持偏振发射

旋转慢

Application
  • Immunoassay
example
XMUGXQ PFS0801Example

Ab + Ag-F  Ab:Ag-F

异硫氰酸荧光素(FITC)标记的paclitaxel(一种具有抗癌活性的物质)与单克隆paclitaxel 抗体(anti-pactaxel)特异性结合导致荧光偏振增强

example1
XMUGXQ PFS0801Example

Ab:Ag-F + Ag  Ab:Ag + Ag-F

在固定标记抗原(FITC-paclitaxel)和抗体(anti-pactaxel)的条件下,加入待测抗原(非标记抗原paclitaxel),后者与标记抗原竞争抗体,导致荧光偏振下降,由竞争反应与非竞争反应荧光偏振的比值(B/B0)给出测量paclitaxel的标准工作曲线

choosing fluorophore
XMUGXQ PFS0801Choosing fluorophore

Ag-F depolarization

(Ag-F) << 