XMUGXQ PFS0801 Principles of Fluorescence Spectroscopy Chemistry Department XMU

XMUGXQ PFS0801 Chapter Eight Measurement of Fluorescence Anisotropy

XMUGXQ PFS Measurement of AnisotropyMeasurement of Anisotropy 8.2 Polarization Spectra of FluorophoresPolarization Spectra of Fluorophores 8.3 Perrin EquationPerrin Equation 8.4 Time-Dependent Decays of AnisotropyTime-Dependent Decays of Anisotropy 8.5 Nonsymmetric FluorophoresNonsymmetric Fluorophores 8.6 Application of Anisotropy MeasurementApplication of Anisotropy Measurement Content

XMUGXQ PFS Measurement of Anisotropy L-format or single-channel method I VV = kS V I  I VH = kS H I  k 是与荧光量子产率及 仪器因素相关的比例 常数。

XMUGXQ PFS0801 Measurement of Anisotropy Measurement of G factor

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

XMUGXQ PFS0801 Measurement of Anisotropy Excited horizontally DET R H = G ‖ /G  R V /R H = I ‖ /I  

XMUGXQ PFS Polarization Spectra of Fluorophores For a dilute vitrified solution in which fluorophores are random oriented  L A  A E Anisotropy

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.

XMUGXQ PFS0801 Polarization excitation spectrum Polarization spectrum of perylene in propylene glycol ( 丙烯二醇） at -50ºC  (º) r0r0 P0P

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

XMUGXQ PFS0801 Polarization Emission spectrum hv ex hv em AF Generally, the anisotropy is independent of the emission wavelength.

XMUGXQ PFS Perrin equation In the presence of rotational diffusion  A E E  On excitation, the time-resolved decay of anisotropy for a spherical molecule is a single exponential: , rotational correlation time of the fluorophore

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

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

XMUGXQ PFS0801 Perrin equation The alternative forms of perrin equation 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  。

XMUGXQ PFS Time-dependent decays of anisotropy For non-symmetric fluoropholres

XMUGXQ PFS0801 Measurement of time-resolved anisotropy Pulsed excitation method

XMUGXQ PFS0801 Time dependence of fluorescence depolarization

XMUGXQ PFS0801 Measurement of time-resolved anisotropy Modulated excitation method

XMUGXQ PFS0801 Measurement of time-resolved anisotropy

XMUGXQ PFS Nonsymmetric Fluorophores Hindered fluorophores

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  = r 0 (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。

XMUGXQ PFS0801 Nonsymmetric Fluorophores Different fluorophores or fluorophore in different microenvironment For a protein-bound fluorophore r 01 = r 02 If lifetime not change on labeling  1 <<  2

XMUGXQ PFS Application of Anisotropy Measurement Estimation of the micro-viscosities of cell membranes

XMUGXQ PFS0801 Estimation of the micro-viscosities of cell membranes

XMUGXQ PFS0801 Application Estimate the rotational diffusion and volume of protein

XMUGXQ PFS0801 Application Measurement of association reactions r F, in the absence of L r B, at a high protein concentration, all L bind to protein r [P][P] rFrF rBrB K dissociation constant

XMUGXQ PFS0801 Application Denature of DNA r [DNA] r T TCTC

XMUGXQ PFS0801 Application Immunoassay 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

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

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/B 0 ）给出测量 paclitaxel 的标准工作 曲线

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