Principles of Fluorescence Spectroscopy

1. XMUPFS01-ITF02 Principles of Fluorescence Spectroscopy Chemistry Department XMU

2. 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 Fluorophore • 1.6 Fluorescence quenching • 1.7 Resonance energy transfer • 1.8 Time scale of Fluorescence • 1.9 Intensity and Concentration • 1.10 Fluorophore

3. XMUPFS01-ITF02 1.5 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

4. XMUPFS01-ITF02 Absorption and emission spectra

5. XMUPFS01-ITF02 F F   Excitation and emission spectra Excitation spectrum Emission spectra em 3 1 2 4

6. XMUPFS01-ITF02 Three dimension spectrum

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

8. XMUPFS01-ITF02 1.5.5 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

9. XMUPFS01-ITF02 S1 relaxation (10-12 s) S1 hvA hvF  knr S0 Expression Simplified Jablonski diagram : Emission rate Knr: nonradiative decay rate Lifetime Intrinsic or natural lifetime The lifetime of the fluorophore in the absence of nonradiative processes is called the intrinsic lifetime.

10. XMUPFS01-ITF02 1.5.6 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

11. XMUPFS01-ITF02 F  Comparison with a standard Determination 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 5. Repeat 2-4th steps with the sample, and get Ax and Fx

12. XMUPFS01-ITF02 Choose Standards Enough absorbance both standard and sample at chosen excited wavelength. Moderate quantum yield. Examples 1. 0.05 mol / L sulfate of quinine,  = 0.55 2. RuPy3Cl2, deoxygenated solution, 20C,  = 0.042

13. XMUPFS01-ITF02 E H 1.5.7 Fluorescence Anisotropy and Fluorescence Polarization • 光的性质 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 • 偏振光 • 非偏振光

14. 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 吸收跃迁距 absorption transition moment 发射偶极距 emission dipole moment 发射跃迁距 emission transition moment 吸收跃迁距和发射跃迁距共线 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 A 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。 E

15. XMUPFS01-ITF02 M, E E M a  b Principle of photoselective excitation Fluorophores preferentially absorb photons whose electric vectors are aligned parallel to the transition moment of the fluorophore. 光吸收选择示意 a. 吸收几率∝M，b. 吸收几率∝Mcos2

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

17. 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：

18. 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。 问题的说明 DPH 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。

19. 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 = cos2 sin d 荧光体的几率分布

20. XMUPFS01-ITF02 坐标系中的荧光体 I（，）= cos2 I（，）= sin2 sin2 以z轴对称分布

21. XMUPFS01-ITF02 Magic angle 魔鬼角

22. XMUPFS01-ITF02 A,E A E A   E  E 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  = 0 r = 0.4  > 0 r < 0.4 旋转运动（），环境性质

23. 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

24. XMUPFS01-ITF02 hvA hvF S1 relaxation (10-12 s) S1  knr S0 1.6 Fluorescence quenching • quench Decreases in fluorescence intensity are called quenching • Quencher Other molecules colliding or reaction with fluorophores, causing quench Oxygen, halogens …… • Stern-Volmer equation Q kq[Q] Q

25. XMUPFS01-ITF02 1.7 Fluorescence resonance energy transfer • Energy transfer D, Donor; A, Acceptor • Conditions for energy transfer • Distance R0 Forster distance r distance, A and D • Overlap of spectra, A’s absorption and D’s emission D’s emission A’s absorption • Orientation

26. XMUPFS01-ITF02 S1 relaxation (10-12 s) hvA hvF S1 A(S1)  knr A(S0) S0 1.7 Fluorescence resonance energy transfer • Process of deactivation

27. XMUPFS01-ITF02 1.8 Time scale of molecular process in solution • e.g. Collisional quenching of fluorescence by oxygen Diffusion coefficient (D) of O2 at 25C is 2.5 x 10-5 cm2/s. the average distance (x2) an O2 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. Provide a way probing resolved oxygen • e.g. Fluorophore-solvent interaction during the lifetime of fluorophores probing micro-envirnoment, pH, viscosity, polarity, et al.

28. 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），1125-1127

29. 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, 51-62, (1998.3).

30. XMUPFS01-ITF02 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 1.8 Time scale of molecular process in solution • e.g. Rotation diffusion of protein during the lifetime of fluorophores

31. XMUPFS01-ITF02 1.8 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

32. 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).

33. XMUPFS01-ITF02 1.9 Intensity and concentration Principle of quantitative and qualitative analysis by fluorometry • Qualitative probing parameters: • Quantitative determination

34. XMUPFS01-ITF02 1.10 Intrinsic fluorophore and extrinsic fluorophore • Intrinsic fluorophores are those which occur naturally • Extrinsic fluorophores, fluorescence probes

35. 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 Phenylalanine tyrosine

36. XMUPFS01-ITF02 Protein fluorescence

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

38. XMUPFS01-ITF02 membrane

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

40. XMUPFS01-ITF02 Cofactor 辅酶 • Nicotinamide adenine dinucleotide 烟酰胺腺嘌呤二核苷酸 NADH, fluorescent; NAD+ nonfluorescent Related to enzymatic reaction, probing enzymatic process • 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) As a intrinsic probe to study cell, tissue, and protein

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

42. XMUPFS01-ITF02 Fluorescent indicators Fluorophores whose spectral properties are sensitive to a substance of interest, such as K+, Ca2+, Mg2+, Na+, Cl-, O2 and CO2…….