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Principles of Fluorescence Spectroscopy

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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Principles of Fluorescence Spectroscopy

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  1. XMUGXQ PFS0801 Principles of Fluorescence Spectroscopy Chemistry Department XMU

  2. XMUGXQ PFS0801 Chapter Eight Measurement of Fluorescence Anisotropy

  3. XMUGXQ PFS0801 Content 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

  4. XMUGXQ PFS0801 8.1 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。

  5. XMUGXQ PFS0801 Measurement of Anisotropy Measurement of G factor

  6. XMUGXQ PFS0801 DET DET Measurement of Anisotropy • T-format or two-channel method Excited vertically Gi, gain to i component

  7. XMUGXQ PFS0801 DET DET Measurement of Anisotropy Excited horizontally RH = G‖/G RV/RH = I‖/I  

  8. XMUGXQ PFS0801 L A A E   8.2 Polarization Spectra of Fluorophores • Anisotropy For a dilute vitrified solution in which fluorophores are random oriented

  9. XMUGXQ PFS0801 Polarization 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.

  10. XMUGXQ PFS0801 Polarization excitation spectrum Polarization spectrum of perylene in propylene glycol ( 丙烯二醇）at -50ºC

  11. XMUGXQ PFS0801 Polarization excitation spectrum Polarization spectrum of 9-vinyl anthracene in propylene glycol ( 丙烯二醇）at -50ºC

  12. XMUGXQ PFS0801 hvex hvem A F Polarization Emission spectrum Generally, the anisotropy is independent of the emission wavelength.

  13. 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

  14. XMUGXQ PFS0801 Perrin equation For steady state measurement, the anisotropy is an average of r(t) by F(t). Perrin Equation

  15. XMUGXQ PFS0801 Perrin equation For highly viscous solution or a macro-molecule, Where  , / <<1 Otherwise, When  <<,  / >>1 The observed anisotropy will be zero

  16. XMUGXQ PFS0801 Perrin 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。

  17. XMUGXQ PFS0801 8.4 Time-dependent decays of anisotropy For non-symmetric fluoropholres

  18. XMUGXQ PFS0801 Measurement of time-resolved anisotropy Pulsed excitation method

  19. XMUGXQ PFS0801 Time dependence of fluorescence depolarization

  20. XMUGXQ PFS0801 Measurement of time-resolved anisotropy Modulated excitation method

  21. XMUGXQ PFS0801 Measurement of time-resolved anisotropy

  22. XMUGXQ PFS0801 8.5 Nonsymmetric Fluorophores • Hindered fluorophores

  23. XMUGXQ PFS0801 Nonsymmetric 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。

  24. XMUGXQ PFS0801 Nonsymmetric Fluorophores • Different fluorophores or fluorophore in different microenvironment For a protein-bound fluorophore r01 = r02 If lifetime not change on labeling 1 << 2

  25. XMUGXQ PFS0801 8.6 Application of Anisotropy Measurement • Estimation of the micro-viscosities of cell membranes

  26. XMUGXQ PFS0801 Estimation of the micro-viscosities of cell membranes

  27. XMUGXQ PFS0801 Application • Estimate the rotational diffusion and volume of protein

  28. 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

  29. XMUGXQ PFS0801 r r [DNA] T Application • Denature of DNA TC

  30. 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

  31. XMUGXQ PFS0801 Example 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

  32. XMUGXQ PFS0801 Example 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

  33. XMUGXQ PFS0801 Choosing fluorophore Ag-F depolarization (Ag-F) << 

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