Fluorescence Spectroscopy英文文献.pdfVIP

AP!I’ER 15 Fluorescence Spectroscopy Paul G. krley 1. Introduction 1.1. Mechanisms of FZuorescence When a substance absorbs light, its molecules are excited from a ground state to a higher energy level. The molecule can then return to the ground state by losing energy, mainly as heat to its surroundings. In some cases,part of the energy absorbed can be reemitted as radia- tion, usually of a longer wavelength than the exciting light. This pro- cess is known as fluorescence (1). How the light is absorbed and emitted during the fluorescence pro- cessis shown in Fig. 1.The electronic statesof the system arerepresented asA, the ground state, and B, the first excited state. Each electronic state can exist in different vibrational energy levels shown here by lines 0, 1, 2, and so on. Following the absorption of light, electronic excitation occurs usu- ally to a higher vibrational level of B. At this stage if the substance is not fluorescent, the energy will be lost asheat asthe molecule returns to the ground state. If, however, the molecule is fluorescent (i.e., con- tains a fluorophore), then some energy will be lost allowing the mol- ecule to reach the lowest vibrational level of the excited state. Return to the ground statewould then beachieved by reradiation of the absorbed energy as fluorescence. Fluorescence emission has various characteristics of which there are usually few exceptions: From* Methods in Molecular Biology, Vol. 22. Microscopy, Optrcal Spectroscopy, and Macroscoprc Techniques Edited by: C. Jones, B. Mulloy, and A H Thomas Copyright 01994 H