XMUPFS01-ITF02 Principles of Fluorescence Spectroscopy Chemistry Department XMU

XMUPFS01-ITF02 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 1.5 Characteristics of FluorophoreCharacteristics of Fluorophore 1.6 Fluorescence quenchingFluorescence quenching 1.7 Resonance energy transferResonance energy transfer 1.8 Time scale of FluorescenceTime scale of Fluorescence 1.9 Intensity and ConcentrationIntensity and Concentration 1.10 FluorophoreFluorophore

XMUPFS01-ITF Characteristics of fluorophore  Excitation wavelength 激发波长 exExcitation wavelength  Emission wavelength 发射波长 em  Extinction (absorption) coefficient 吸光系数   Stokes’ shift  Fluorescence lifetime 荧光寿命 Fluorescence lifetime  Fluorescence quantum yield 荧光量子产率 Fluorescence quantum yield  Fluorescence Anisotropy 各项异性 rFluorescence Anisotropy  15.8 Fluorescence Polarization 荧光偏振 p

XMUPFS01-ITF02 Absorption and emission spectra

XMUPFS01-ITF02 Excitation and emission spectra Excitation spectrumEmission spectra F F em

XMUPFS01-ITF02 Three dimension spectrum

XMUPFS01-ITF02 Three dimension spectra 引自林竹光 等人的论文 ex / nm em / nm

XMUPFS01-ITF Fluorescence lifetime 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

XMUPFS01-ITF02 Expression relaxation ( s) S0S0 S1S1 S1S1 hv A hv F  k nr Simplified Jablonski diagram  : Emission rate K nr : nonradiative decay rate LifetimeIntrinsic or natural lifetime The lifetime of the fluorophore in the absence of nonradiative processes is called the intrinsic lifetime.

XMUPFS01-ITF fluorescence quantum yield Definition The fluorescence quantum yield is the ratio of the number of photons emitted to the number absorbed Expression Relationship with lifetime

XMUPFS01-ITF02 Determination Comparison with a standard 1. Chose a Standard with a quantum yield  s 2. At I determine the absorption (A s ) 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 F s F 5. Repeat 2-4th steps with the sample, and get A x and F x

XMUPFS01-ITF02 Choose Standards Enough absorbance both standard and sample at chosen excited wavelength. Moderate quantum yield mol / L sulfate of quinine,  = 0.55 Examples 2. RuPy 3 Cl 2, deoxygenated solution, 20  C,  = 0.042

XMUPFS01-ITF 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 E H 偏振光 非偏振光

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 吸收跃迁距和发射跃迁距共线  A E 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

XMUPFS01-ITF02 Principle of photoselective excitation Fluorophores preferentially absorb photons whose electric vectors are aligned parallel to the transition moment of the fluorophore. EM, E ba  M 光吸收选择示意 a. 吸收几率∝ M ， b. 吸收几率∝ Mcos 2 

XMUPFS01-ITF02 z 激发偏振器 发射偏振器 x y 检测器 II I  Definition polarization anisotropy For fluorophore

XMUPFS01-ITF02 Polarization and anisotropy 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：

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。 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

XMUPFS01-ITF02 Isotropic 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  = cos 2  sin  d  荧光体的几率分布

XMUPFS01-ITF02 坐标系中的荧光体 I  （  ，  ） = cos 2  I  （  ，  ） = sin 2  sin 2  以 z 轴对称分布

XMUPFS01-ITF02 Magic angle 魔鬼角

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 旋转运动（  ），环境性质  = 0  > 0  A E E 

XMUPFS01-ITF02 Losing of anisotropy In fluid solution, most fluorophores roatate extensively in 50 – 100 ps. 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? Perrin equation

XMUPFS01-ITF Fluorescence quenching quench Decreases in fluorescence intensity are called quenching Quencher Stern-Volmer equation relaxation ( s) S0S0 S1S1 S1S1 hv A hv F  k nr Q Q k q [Q] Other molecules colliding or reaction with fluorophores, causing quench Oxygen, halogens ……

XMUPFS01-ITF Fluorescence resonance energy transfer Energy transfer Conditions for energy transfer  DistanceR 0 Forster distance r distance, A and D  Overlap of spectra, A’s absorption and D’s emission D’s emissionA’s absorption D, Donor; A, Acceptor  Orientation

XMUPFS01-ITF Fluorescence resonance energy transfer relaxation ( s) S0S0 S1S1 S1S1 hv A hv F  k nr A(S 0 ) A(S 1 ) Process of deactivation

XMUPFS01-ITF Time scale of molecular process in solution e.g. Collisional quenching of fluorescence by oxygen Diffusion coefficient (D) of O 2 at 25  C is 2.5 x cm 2 /s. the average distance (  x 2 ) an O 2 can diffuse in 10 ns given by the Einstein equation, 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

XMUPFS01-ITF02 Probing pH 马丽华，温珍昌，孙向英，江云宝，高等学校化学学报， 2001 ， 22 （ 7 ），

XMUPFS01-ITF02 Probing polarity ● 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, , (1998.3).

XMUPFS01-ITF 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

XMUPFS01-ITF Time scale of molecular process in solution 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

XMUPFS01-ITF02 Structure of DPPG ● 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).

XMUPFS01-ITF Intensity and concentration Principle of quantitative and qualitative analysis by fluorometry Qualitative probing parameters: Quantitative determination

XMUPFS01-ITF Intrinsic fluorophore and extrinsic fluorophore Intrinsic fluorophores are those which occur naturally Extrinsic fluorophores, fluorescence probes

XMUPFS01-ITF02 Protein fluorescence Indole group of tryptophan 色氨酸中的吲哚基团 ex = 280, em = 340, highly sensitive to solvent polarity tyrosinePhenylalanine

XMUPFS01-ITF02 Protein fluorescence

XMUPFS01-ITF02 membrane Typically do not display intrinsic fluorescence Extrinsic membrane fluorescence probe DPH Rhodamine B

XMUPFS01-ITF02 membrane

XMUPFS01-ITF02 DNA DNA is weakly fluorescent or nonfluorescent Extrinsic fluorescence probe Acridine orangeEthidium bromide Staining of cells with dyes that bind to DNA is widely used to visualize and identify chromosomes.

XMUPFS01-ITF02 Cofactor 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

XMUPFS01-ITF02 Other fluorophores Fluorescein Dansyl chloride 丹磺酰氯 Chlorophyll 叶绿素

XMUPFS01-ITF02 Fluorescent indicators Fluorophores whose spectral properties are sensitive to a substance of interest, such as K +, Ca 2+, Mg 2+, Na+, Cl -, O 2 and CO 2 …….