Basics of fluorescence

Abstract

The objective of this chapter is to provide a simple understanding of molecular spectroscopy, with a focus on concepts and techniques relevant to microscopy. The inherently quantum mechanical concepts of phenomena, including energy levels, radiative and nonradiative processes, and quantum yields, will be developed from a nonmathematical framework that is often used by experts in spectroscopy when discussing general principles. This simplified framework is developed into a practical and useful understanding of optical spectroscopy aimed at nonspecialists. This groundwork supports the more detailed discussion of complex techniques in the following chapters. To begin the chapter we will outline basic features and properties of light and energy states of molecules. We then connect the two by exploring the interaction of light with molecules, initially for an isolated molecule. We will consider the key properties of absorption and emission and also those properties that are the bane of microscopy—the so called nonradiative mechanisms. After considering the isolated molecule, we will discuss interactions of molecules with their environment, which develops the concepts underpinning photobleaching and quenching, and also the more recent techniques of FRET and FLIM. En route we will examine the excitation and emission spectra of typical dyes, which can be found in any dye catalogue (see also Jameson et al. [1] for further reading).