【课件】荧光光谱方法及原理剖析.ppt

荧光光谱 参考书 赵南明，周海梦. 生物物理学，高等教育出版社，2000 Emission Spectroscopy, Chapter 11, in K.E. van Holde, W.C. Johnson and P.S. Ho eds. Principles of Physical Biochemistry, Prentice-Hall, 1998 荧光光谱的特点 荧光光谱灵敏度高的原因 荧光辐射的波长比激发光波长长，测量到的荧光频率与入射光的频率不同； 荧光在各个方向上都有发射，因此可以在与入射光成直角的方向上检测； 这样，荧光不受来自激发光的本底的干扰，灵敏度大大高于紫外-可见吸收光谱，测量用的样品量很少，且测量方法简便。 基本原理 Fluorescence Fluorescence is emission from a singlet state, where the electron spins in the molecule are paired. Fluorescence excitation by absorption of energy loses energy as heat by internal conversion the molecule hesitates at the lowest vibrational level of the first singlet, because a relatively large amount of energy must be lost to go to the ground state. during this hesitation one of three things may happen: the molecule may fluoresce it may convert to the triplet (intersystem crossing) it may go to the ground state without emitting a photon (a nonradiative transition). EMISSlON LlFETlMES 仪器结构 FLUORESCENCE INSTRUMENTATlON Fluorophores are compared using the ratio of their fluorescence yields Often fluorophores are compared using the ratio of their fluorescence yields. As we see in Figure 11.13, this relative fluorescence yield will depend on the wavelength chosen for observation because of the shapes of the fluorescence spectra. Additivity properties of polarization the average anisotropy measured for a mixture of components is just the sum of the individual anisotropies weighed by their fluorescence yield F, (11.14) 环境对荧光参数的影响 Solvent effects Solvent effects will affect the position of the fluorescence band. The effects of a solvent on fluorescence can be very large, and many studies of macromolecules use this fact. General solvent effects depend on the polarizability of the solvent, and increasing the dielectric constant usually shifts the fluorescence to longer wavelength. Specific solvent effects are the result of chemical reaction of the excited state