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
某些物质射出波长比入射光长的光，如果这个时间比 较短，这种光就称为荧光。荧光由一种能发荧光的
矿物 萤石(fluospar)而得名。
我们这里要介绍的荧光，是指物质在吸收紫外 光和可见光后发出的波长较长的紫外荧光或可见荧 光。
除了紫外光和可见光可能激发荧光外，其它的
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。
荧光光谱第二个特点是信息量较大
荧光光谱能提供较多的参数，例如激发谱、发射谱、峰位、峰 强度、荧光寿命、荧光偏振度等。 荧光光谱还可以检测一些紫外-可见吸收光谱检测不到的时间过 程过程。紫外和可见荧光涉及的是电子能级之间的跃迁，荧光 产生包括两个过程：吸收以及随之而来的发射。每个过程发生 的时间与跃迁频率的倒数是同一时间量级(大约10-15秒)，但两 个过程中有一个时间延搁，大约为10-8秒，这段时间内分子处 于激发态。激发态的寿命取决于辐射与非辐射之间的竞争。由 于荧光有一定的寿命，因此可以检测一些时间过程与其寿命相 当的过程。例如，生色团及其环境的变化过程在紫外可见吸收 的10-15秒的过程中基本上是静止不变的，因此无法用紫外可见 吸收光谱检测，但可以用荧光光谱检测。
荧光光谱的特点
Fluorescence is a sensitive technique for detecting biological materials
Barak and Webb demonstrated that as few as 30 fluorescent dye molecules bound to low density lipoprotein molecules can be detected on cell surfaces by microscopy using a sensitive video camera. With even more sophisticated methods others have moved toward detection of single fluorophore molecules in a laserexcited flow stream. Because this sensitivity can combine with ease of use and the potential for multiparameter analysis, fluorescence has become widely used in biology and medicine.
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).
光如红外光、X射线也可能激发出荧光，因此除紫外 荧光或可见荧光外，还有红外荧光、X射线荧光等。
In discussing absorption we have been concerned entirely with the excitation of a molecule from its ground state to a higher energy level and have given no consideration to the subsequent fate of the excited molecule. In many cases the sequel is not very interesting; the energy is transferred as heat to the surroundings. In some cases light is emitted by the sample either as fluorescence or phosphorescence. We will discuss the general characteristics of emission and ignore the exceptions.
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