Introduction to Fluorescence Techniques - UCCS （介绍荧光技术ucc）.pdfVIP

Introduction to Fluorescence Techniques Fluorescent probes enable researchers to detect particular photons to be detected against a low background, isolated from components of complex biomolecular assemblies, including live excitation photons. In contrast, absorption spectrophotometry cells, with exquisite sensitivity and selectivity. The purpose of requires measurement of transmitted light relative to high inci- this introduction is to briefly outline fluorescence techniques for dent light levels at the same wavelength. newcomers to the field. Fluorescence Spectra The entire fluorescence process is cyclical. Unless the fluoro- The Fluorescence Process phore is irreversibly destroyed in the excited state (an important Fluorescence is the result of a three-stage process that occurs phenomenon known as photobleaching, see below), the same in certain molecules (generally polyaromatic hydrocarbons or fluorophore can be repeatedly excited and detected. The fact that heterocycles) called fluorophores or fluorescent dyes. A fluores- a single fluorophore can generate many thousands of detectable cent probe is a fluorophore designed to localize within a specific photons is fundamental to the high sensitivity of fluorescence region of a biological specimen or to respond to a specific stimu- detection techniques. For polyatomic molecules in solution, the lus. The process responsible for the fluorescence of fluorescent discrete electronic transitions represented by hυEX and hυEM in probes and other fluorophores is illustrated by t