Fluorescence Spectroscopy And Microscopy

Reflecting the expanding field's need for reliable protocols, Fluorescence Spectroscopy and Microscopy: Methods and Protocols offers techniques from a worldwide team of experts on this versatile and vital subject. The topics covered fall into four broad categories: steady-state fluorescence spectroscopy, time-resolved fluorescence spectroscopy, fluorescent probe development, and the various sub-categories of fluorescence microscopy, such as fluorescence recovery after photobleaching (FRAP), live cell FRET imaging (FRETim), fluorescence lifetime imaging (FLIM), fluorescence fluctuation spectroscopy (FFS), and single-molecule fluorescence spectroscopy (smFS). Written as a part of the popular Methods in Molecular Biology series, chapters include the kind of unambiguous detail and key implementation advice that proves essential for successful results.

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.

Providing much-needed information on fluorescence spectroscopy and microscopy, this ready reference covers detection techniques, data registration, and the use of spectroscopic tools, as well as new techniques for improving the resolution of optical microscopy below the resolution gap. Starting with the basic principles, the book goes on to treat fluorophores and labeling, single-molecule fluorescence spectroscopy and enzymatics, as well as excited state energy transfer, and super-resolution fluorescence imaging. Examples show how each technique can help in obtaining detailed and refined information from individual molecular systems.

This first volume in the new Springer Series on Fluorescence brings together fundamental and applied research from this highly interdisciplinary and field, ranging from chemistry and physics to biology and medicine. Special attention is given to supramolecular systems, sensor applications, confocal microscopy and protein-protein interactions. This carefully edited collection of articles is an invaluable tool for practitioners and novices.

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.

During the past two decades, there has been an increasing appreciation of the significant value that lifetime-based techniques can add to biomedical studies and applications of fluorescence. Bringing together perspectives of different research communities, Fluorescence Lifetime Spectroscopy and Imaging: Principles and Applications in Biomedical Diagnostics explores the remarkable advances in time-resolved fluorescence techniques and their role in a wide range of biological and clinical applications. Broadly accessible, the book captures the state-of-the-art of fluorescence lifetime metrology and imaging and provides current perspectives on their applications to biomedical studies of intact tissues and medical diagnosis. The text introduces these techniques within the wider context of fluorescence spectroscopy and describes basic principles underlying current instrumentation for fluorescence lifetime imaging and metrology (FLIM). It also covers the wide range of methods, including single channel (point) spectroscopy, fluorescence lifetime imaging microscopy, and single- and multi-photon excitation. Edited by pioneers in this field, with contributions from leading experts, the book includes an overview of complementary techniques that help researchers beginning FLIM research. It offers a comprehensive treatment of fundamental principles, instrumentation, analytical methods, and applications. It also provides an overview of the label-free contrast available from lifetime measurements of tissue autofluorescence and the prospects for exploiting this for clinical applications and biomedical research including drug discovery.

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.

The increased use of fluorescence techniques is greatly enhanced by the improved instrumentation pioneered by inventive scientists and now made available commercially by several high-tech companies. Moreover, the design and development of many new molecular probes with higher selectivity for specific microenvironmental properties has stimulated many new researchers to employ fluorescence techniques for solving their problems. This topic book, the second in his series, reflects this exciting scientific progress and deals, among others, with new approaches and new probes in fluorescence spectroscopy, single molecule fluorescence, applications in biomembrane and enzyme studies and imaging of living cells.