Fluorescence Spectroscopy In Biology

Volume 3 of this new series focuses on brandnew research and applications in biology, biophysics and other fields of life sciences. Many frontline researcher have contributed to this highly attractive and interdisciplinary volume which spans the entire field of present fluorescence spectroscopy including nanotechnology, membrane and DNA studies and fluorescence imaging in cancer research.

At the time that the editors conceived the idea of trying to organize the meeting on which the contents of this volume are based and which became, in March 1980, a NATO Advanced Study Institute, the techniques of time-resolved fluorescence spectroscopy, in both the nanosecond and sub-nanosecond time-domains, might reasonably have been said to be coming of age, both in their execution and in the analysis and interpretation of the results obtained. These techniques, then as now, comprised mainly a number of pulse methods using laser, flash-lamp or, most recently, synchrotron radiation. In addition, significant developments in the more classical phase approach had also rendered that method popular, utilizing either modulation of an otherwise continuous source or, again recently, the ultra-rapid pulse rate attainable with a synchrotron source. In general terms, time-resolved fluorescence studies are capable, under appropriate conditions, of supplying direct kinetic information on both photophysics and various aspects of molecular, macromolecular and supramolecular structure and dynamics. The nanosecond and sub-nanosecond time-scales directly probed render these techniques particularly appropriate in studying relaxation and fluctuation processes in macromolecules, particularly biopolymers (e. g. proteins, nucleic acids), in supramolecular assemblies such as cell membranes, and in a variety of relatively simpler model systems.

This book provides the reader with an updated comprehensive view of the rapidly developing and fascinating field of fluorescence spectroscopy and microscopy. In recent years, fluorescence spectroscopy and microscopy have experienced rapid technological development, which has enabled the detection and monitoring of single molecules with high spatial and temporal resolution. Thanks to these developments, fluorescence has become an even more popular method in physical, biological and related fields. This book guides the reader through both basic and advanced fluorescence spectroscopy and microscopy approaches with a focus on their applications in membrane and protein biophysics. Each of the four parts: A - Fluorescence Spectroscopy, B - Fluorescence Microscopy, C - Applications of Fluorescence Spectroscopy and Microscopy to biological membranes and D - Applications of Fluorescence Spectroscopy to protein studies are written by experts within the field. The book is intended for both complete beginners who want to quickly orient themselves in the large number of existing fluorescent methods, as well as for advanced readers who are interested in particular methods and their proper use.

Fluorescence spectroscopy is an important investigational tool in many areas of analytical science, due to its extremely high sensitivity and selectivity. With many uses across a broad range of chemical, biochemical and medical research, it has become an essential investigational technique allowing detailed, real-time observation of the structure and dynamics of intact biological systems with extremely high resolution. It is particularly heavily used in the pharmaceutical industry where it has almost completely replaced radiochemical labelling. Principles and Applications of Fluorescence Spectroscopy gives the student and new user the essential information to help them to understand and use the technique confidently in their research. By integrating the treatment of absorption and fluorescence, the student is shown how fluorescence phenomena arise and how these can be used to probe a range of analytical problems. A key element of the book is the inclusion of practical laboratory experiments that illustrate the fundamental points and applications of the technique.

Fluorescence spectroscopy has traditionally found wide application in bio chemistry and cell biology. Since there are relatively few naturally occurring fluorescent biomolecules, fluorescence spectroscopy offers a combination of great specificity and sensitivity. Historically, these features have been ex ploited with great success utilizing both intrinsic and extrinsic probes. Re cent applications have built upon these traditional strengths and have re sulted in the development of new instrumental techniques, novel and convenient fluorescent probes, and a deeper, theoretical understanding of fundamental processes. Frequently, fluorescence techniques are tailored to attack a specific biological problem. These new methods in turn produce new physical situations and phenomena which are often of interest to the physical chemist. Thus, progress in one area stimulates renewed interest in other areas. The goal of this book is to provide detailed monographs on the use of fluorescence to investigate problems at the forefront of biochemistry and cell biology. This book is not meant to be a comprehensive survey but rather to highlight areas of recent developments. It is designed to be readable to the novice and yet provide sufficient detail for the expert to keep abreast of recent developments. The book is organized so that it proceeds from simple biochemical sys tems to more complex cell biological ones. Chapter I on fluorescence quenching of biological structures is a good introductory chapter. It intro duces a number of elementary concepts and discusses applications to pro teins and biomembranes.

Fluorescence methods are being used increasingly in biochemical, medical, and chemical research. This is because of the inherent sensitivity of this technique. and the favorable time scale of the phenomenon of fluorescence. 8 Fluorescence emission occurs about 10- sec (10 nsec) after light absorp tion. During this period of time a wide range of molecular processes can occur, and these can effect the spectral characteristics of the fluorescent compound. This combination of sensitivity and a favorable time scale allows fluorescence methods to be generally useful for studies of proteins and membranes and their interactions with other macromolecules. This book describes the fundamental aspects of fluorescence. and the biochemical applications of this methodology. Each chapter starts with the -theoreticalbasis of each phenomenon of fluorescence, followed by examples which illustrate the use of the phenomenon in the study of biochemical problems. The book contains numerous figures. It is felt that such graphical presentations contribute to pleasurable reading and increased understand ing. Separate chapters are devoted to fluorescence polarization, lifetimes, quenching, energy transfer, solvent effects, and excited state reactions. To enhance the usefulness of this work as a textbook, problems are included which illustrate the concepts described in each chapter. Furthermore, a separate chapter is devoted to the instrumentation used in fluorescence spectroscopy. This chapter will be especially valuable for those perform ing or contemplating fluorescence measurements. Such measurements are easily compromised by failure to consider a number of simple principles.