Introduction to Fluorescence and its Application to Membrane Structure and Dynamics
This chapter presents an overview of the molecular basis of fluorescence spectroscopy and a physical description of the fluorescence-based processes that can be used to probe the cell surface. The absorption and emission of visible or ultraviolet light involves transitions between the ground and electronic excited states of a chromophore, usually consisting of a delocalized p–system. The probability of fluorescence emission, just like that of absorption, is given by the square of the transition moment. Since the probability of excitation depends on the square of the transition moment, absorption of polarized light by a rigid and well-oriented sample, is characterized by a cosine-squared angular dependence. Coupling fluorescence excitation and detection to a microscope allows the application of the aforementioned techniques to the membranes of individual cells. Since fluorescence can, in principle, be obtained from single molecules, fluorescence microscopy does indirectly provide molecular resolution.