Fluorescence: Basic Concepts, Practical Aspects, and Some Anecdotes

Abstract

The theoretical foundations of fluorescence spectroscopy were established in the first half of the twentieth century by pioneers including Enrique Gaviola, Jean and Francis Perrin (father and son), Peter Pringsheim, Sergei Vavilov, F. Weigert, F. Dushinsky, Alexander Jabloński, Theodor Förster, and, more recently, Gregorio Weber. In the last quarter of the twentieth century, advances in electronics, lasers, computers, and molecular biology have allowed fluorescence methodologies to assume an important role in diverse disciplines including chemistry, cell biology, and the biomedical sciences. This volume of 'Methods in Enzymology' covers many of the most exciting new developments in fluorescence spectroscopy developments and techniques that presently define the state of the art. In this article, however, we wish to remind readers of the origins of several important aspects of fluorescence spectroscopy. We also wish to discuss some practical aspects of fluorescence determinations which are sometimes forgotten as those new to these methods often focus on learning the software associated with commercial instrumentation. Much of the modem 'point-and-click' software approach allows the novice to immediately apply fluorescence methods to their particular research problems, taking advantage of the highly sophisticated instrumentation and probe chemistries that are now readily available. However, kits and user friendly software should not dissuade beginners from learning the fundamentals of fluorescence methodologies and instrumentation. Such knowledge not only allows avoidance of potential pitfalls, recognition of artifacts, and fuller appreciation of the applicability of fluorescence techniques, it also makes the research more interesting and fun!