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4. 荧光偏振

If linear polarized light is used to excite an ensemble of fluorophores, only those fluorophores aligned with the plane of polarization will be excited. There are 2 scenarios for the emission.

Perrin Weber Dandliker Jolley

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荧光偏振度和各向异性•

荧光偏振与荧光体的分子形状、转动速度；与荧光体的吸

光对偏振激发的取向、光选择性、激发矩与发射矩是否共 线等因素有关。

•

P和A的测量可揭示荧光体吸收光子和随后发射光子的平 均角移。

I// -I⊥

I// +I⊥

P=

I// -I⊥

I// +2I⊥

A=

荧光偏振度Fluorescence polarization --p荧光各向异性Fluorescence anisotropy – A or r

P = 3 A / (2 + A)P = 3 A / (2 + A)A = 2 P / (3 A = 2 P / (3 –– P)P)

The fluorescence anisotropy of a fluorophore reflects the molecule’s ability to rotate in its microenvironment, which depends on the viscosity of the solution, the fluorescence lifetime of the fluorophore, and the size and mass of the molecule to which the fluorophore is attached.

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(1) 荧光偏振度的物理意义：

A ：I//

=I⊥

，P=0，自然光，荧光分子运动很 快，取向随机。（稀溶液）

B：I//

或I⊥

为0 ，P=±1 偏振光，荧光分子运 动很慢，取向有序。

C：

I//

≠I⊥

≠

0 ，0<P<1，生物大分子的荧光 属于这种情况。

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Fluorescence polarization (FP) can be considered a competition between the molecular motion and the lifetime of fluorophores in solution.

θrot is the rotational relaxation (correlation) time (the time required to rotate through an angle whose cosine is 1/e, or approximately 68.5°) τfl is the fluorescence lifetime of the excited fluorescent probe

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Physical basis of fluorescence polarization assays. Dye molecules with their absorption transition vectors (arrows) aligned parallel to the electric vector of linearly polarized light (along the vertical page axis) are selectively excited. For dyes attached to small, rapidly rotating molecules, the initially photoselectedorientational distribution becomes randomized prior to emission, resulting inlow fluorescence polarization. Conversely, binding of the low molecular weight tracer to a large, slowly rotating molecule results in high fluorescence polarization. Fluorescence polarization therefore provides a direct readout of the extent of tracer binding to proteins, nucleic acids and other biopolymers.

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(2) 环境因素及分子运动对荧光偏振度的影响

A .温度的影响：

溶液粘度

A：温度的影响

温度升高，P降低B：溶液粘度

粘度升高，P升高

C：分子的运动—转动

旋转弛豫时间rotational relaxation time — ρ

V: the molecular volume of the molecule

η: the viscosity of the medium

τ: the fluorescence lifetime of the fluorophore

T: the temperature

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影响荧光偏振的因素

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(3) 荧光偏振测量

Steady-State Fluorescence PolarizationTime-Resolved Fluorescence Polarization

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稳态荧光偏振

The Perrin equation (1926)(1/P)-(1/3)=((1/P0)-(1/3))(1+(τ/θ))r = r0 / (1 + (τ/ θ))The Stokes equation (for spherically symmetrical molecules)θ =ηV / RTr = r0 / (1 + (τRT /ηV))

Corresponding expressions for spherically unsymmetrical and ellipsoidal molecules can be found in literatures.

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时间分辨荧光偏振

Following a pulse excitation, the fluorescence anisotropy of a spherical particle in a homogeneous isotropic medium decays exponentiallyr = r0 exp ( –t /θ) Non-spherical particles (most biopolymer): complexChromophore (or a molecular domain) rotates around the bond linking it to the biopolymer: complexAnisotropic environment (like in phospholipidbilayers): complex

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Application

Fluorescence polarization measurements have long been a valuable biophysical research tool for investigating processes such as membrane lipid mobility, myosin reorientation and protein–proteininteractions at the molecular level.Immunoassays that have been developed and used extensively for clinical diagnostics represent the largest group of bioanalytical applications. The more recent advent of microplate readers equipped with polarizing optics has led to the adoption of fluorescence polarization as a readout mode for high-throughput screening

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ApplicationImmunoassay

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

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Example Ab + Ag-F → Ab:Ag-F

异硫氰酸荧光素

(FITC) 标 记 的

paclitaxel 与 单 克

隆paclitaxel 抗体

(anti-pactaxel) 特

异性结合导致荧光

偏振增强

紫杉醇

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1.3 激发光谱和荧光光谱

任何荧光化合物都具有两种特征 光谱：

•荧光激发光谱（吸收光谱）

—固定某一发射波长，测定该波 长下的荧光发射强度随激发波长 变化所得的光谱。

•荧光发射光谱（荧光光谱）— —固定某一激发波长，测定荧光

发射强度随发射波长变化得到的 光谱。

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荧光光谱的特点：

①斯托克斯位移（Stokes shift)：是

相同电子跃迁在吸收光谱和发射光 谱（如荧光光谱和拉曼光谱）中最 强波长间的差值。是一个表示分子 发光特性的物理常数，它表示分子 在回到基态以前，在激发态寿命期 间能量的消耗。

②荧光光谱与激发波长无关。

③吸收光谱与发射光谱大致成镜像对称。

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1.4 Fluorescence Quenching

•Internal quenching due to intrinsic structural feature e.g. structural rearrangement.

•External quenching interaction of the excited molecule with another molecule in the sample or absorption of exciting or emitted light by another chromophore in sample.

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猝灭(quenching)

碰撞猝灭荧光物质分子与猝灭分子碰撞，前者损失能量导致荧光强度降低。

转入三重态的猝灭3O2

1 O2

组成化合物的猝灭

荧光物质分子与猝灭分子作用而形成络合物， 这种络合物本身不发荧光；也可能由于其吸收 特性而起内滤光作用。

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发生电子转移反应的猝灭

某些猝灭剂分子与荧光物质分子相互作用 时，发生了电子转移反应，因而引起荧光的

猝灭。

猝灭剂主要包括金属离子，I-，Br-，CNs-等

荧光物质的自猝灭荧光溶液浓度过大时，常发生自猝灭现象。

激发分子在发出荧光之前和为激发分子碰撞造成。

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Stern-Volmer equation

Fluorescence quenching is a function of:The excited state lifetimeThe diffusion-limited quenching constantThe concentration of the quencher

The Stern Volmer slope: KSV = τs kQ

ΦF0/ ΦF = 1 + τs kQ [Q]

Stern-Volmer equation

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1.5 Fluorescence Resonance Energy Transfer

Known as fluorescence resonance energy transfer (FRET) or Förster energy transfer. It is the radiationless transfer of excitation energy from a donor to an acceptor. An important consequence of this transfer is that there is no emission of light by the donor. The acceptor may or may not be fluorescent. FRET is a distance-dependent interaction where the energy transfer occurs typically over a distance of 1-10nm. The distance dependent nature of FRET is highlighted by the fact that it is proportional to the inverse sixth power of the intermolecular separation.

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If the fluorophores (extrinsic or intrinsic) have unique locations within the protein or complex, it is possible for emission light energy from A to be absorbed by B and to be emitted as part of B’s emission spectrum

A/Q

Fluor A

λ2λ1

Absorbanceemission

A/Q

λ (nm)

Fluor B

λ2 λ3

Absorbanceemission

Fluorescence resonance energy transfer (FRET)

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•FRET is dependent on the distance, R, between the two fluors. Used to measure distances in proteins, membranes, & macromolecular assemblies 10 to 80 Å apart.

•Efficiency of the energy transfer (E) from donor to acceptor (A to B) defined by

E = R06

R06 + R6

•R = distance between A & B, R0 is a constant calculated from absorption and emission spectra.

•E can be calculated from fluorescence intensity (F)

E = Fda/FdFda = F in the presence of acceptor, Fd = F in the absence of acceptor.

•Once E is known, R can be calculated from the first equation if R0 is known.

FRET is used as a ‘spectroscopic ruler’

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Limitations

•Limited range (10 – 80 Å) if R is > 8 nm E is very small

•Need 2 fluorophores, a donor & an acceptor.

•Assumptions are made in the calculation of R0 such as the orientation of the donor & the acceptor.

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FRET optical shortcomingsOne major concern of FRET is related to the UV/vis-excitation nature of the energy donors, which normally are fluorescent dyes or proteins or semiconductor quantum dots. In this excitation window, unfortunately, rather strong autofluorescence and scattering light always arise from biomolecules when the assay is conducted in biological sample matrixes. Another issue of FRET assays is the unexpected coexcitation of the energy donor and the acceptor because of the overlap of their excitation spectra, which at present is quite hard to eliminate due to the relatively small Stokes-shift of most down-converting fluorophores.

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光散射常是实验操作中灵敏度的一种限制因素。容器表面的散射光减小狭缝或缩小探测器的窗口，可以减小来自池壁的散射光影响。丁铎尔(Tyndall)散射:样品池中如有胶体颗粒或气泡存在时，和它相遇的入射光就会改变方向进入监测器，这种现象称为丁铎尔(Tyndall)散射。瑞利(Rayleigh)散射:溶质分子吸收了频率较低的光线后，不足以使分子中的电子跃迁到激发态，而只是上升到基态中较高的振动能级，并在极短的时间内返回到原来能级，而释放出与激发光相同的光线，类似于发生了弹性散射几乎没有能量损失，称为瑞利散射。拉曼(Raman)散射:分子吸收了频率较低的光线后，上升到基态中较高的振动能级之后，返回到稍高于原来的能级时产生的，在此过程中消耗了部分能量，类似于发生了非弹性散射，因此释放的光线比激发光波长稍长。

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二、荧光与结构的关系

（1）电子跃迁类型

发射

π*→π跃迁比π*→n跃迁更常见

（2）共轭效应

芳香族化合物的荧光最常见且最强，大多 数未取代芳烃在溶液中发荧光，随着环的数目和稠合程 度增加，荧光峰红移，Φ↑。简单杂环化合物不发荧 光，但具有稠环结构的杂环化合物都发荧光。

（3）平面刚性结构效应

有刚性结构的分子容易发荧光， 刚性和共平面性的增加有利于荧光发射。

联苯

Φ=0.2 芴 Φ=1

CH2

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化合物 相对荧光强度

苯 10

C6 H5 COOHC6 H5 NO2

30

C6 H5 CH3

C6 H5 OHC6 H5 OCH3

C6 H5 NH2

C6 H5 CN

1718202020

C6 H5 ClC6 H5 BrC6 H5 I

750

(4)取代基的影响

芳环上有羧基、羰基或 亚硝基等吸电子基团取 代时，荧光减弱；

给电子取代基如-OH、- NH2

、-CN、-OCH3

等会使 荧光强度增加，荧光波 长长移。

重原子效应

含有重原 子的分子中，系间窜跃 的几率大,使荧光减弱，

磷光增强。

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荧光与环境因素的关系

★温度降低会使荧光强度增大；

★pH 带有酸性或碱性取代基的芳 香化合物的荧光与pH有关；

★溶剂

溶剂极性增加有时会使荧 光强度增加，荧光波长红移； 若溶剂和荧光物质形成氢键或 使荧光物质电离状态改变，会 使荧光强度、荧光波长改变； 含重原子的溶剂（碘乙烷、四 溴化碳）使荧光减弱。

★溶解氧的存在往往使荧光强度降 低。

化合物 相对荧光 强度

C6 H5 OH 18 C6 H5 O— 10C6 H5 NH2 20

+C6 H5 NH3 0

指示剂颜色变化pH范围萘酚无色--黄绿8.2-10.3荧光素浅绿-绿

4.0-5.0

丫啶橙浅黄绿--黄 8.0-10.0

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三、

荧光分析仪器和荧光法的应用

2.1 荧光分光光度计

荧光分光光度计既可用于定量分

析，也可用于测绘激发光谱和荧

光光谱。第一单色器选择激发光

波长（＞250nm的紫外光），故

称为激发单色器。第二单色器

（荧光单色器）与激发光入射方

向垂直，并选择荧光波长，可提

高方法的选择性和准确度。

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Absorption Fluorescence Luminescence