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.

In recent years, the introduction of the combined use of fluorescence spectroscopy and microscopy has opened up exciting new arenas of scientific investigation. The combination of spectroscopic techniques and video microscopy enables acquisition of quantitative data about dynamic processes. It allows the investigation of molecular activity as it occurs--at the level of a single, intact, living cell--and provides extraordinary detail regarding biological organization and function. This new work covers emerging technologies and their applications in a variety of disciplines, including chemistry, biology, physics, computer science, engineering, medicine, and pharmacology. These topics are presented by contributing authors who are leading--and pioneering--researchers in their fields. They offer a critical evaluation of both theoretical and practical aspects of the use of these combined technologies in a way that cuts across disciplines, promoting interactivity and dialogue among scientists in different fields. Each chapter is devoted to a new, unique, or otherwise novel application of the combination of imaging techniques and fluorescence spectroscopy. In addition to covering established methods and instrumentation, the contributors also identify potential new applications in a number of areas. Throughout, they emphasize the multifunctional and multidimensional uses of the marriage of fluorescence imaging spectroscopy and digital imaging microscopy. They show how these technologies can be used in combination to obtain distinct yet complementary information, allowing two- and three-dimensional measurements, as well as manipulations of the sample under study in real time. This book makes the field of fluorescence imaging spectroscopy and microscopy accessible to scientists in a variety of research fields. It is also an excellent text for graduate students, a reference to the latest techniques, and a source of ideas to stimulate future investigation. An exploration of ideas, methods, and applications in the exciting new area of fluorescence imaging spectroscopy and microscopy This book covers the latest in techniques and advances that have developed from the combined use of fluorescence imaging spectroscopy and video microscopy. A relatively new innovation, it has revolutionized molecular research in a wide range of disciplines. This approach allows acquisition of quantitative data regarding real-world biological processes--providing unprecedented insight into the physiology of living cells and their molecular structures and functions. Written by a diverse group of leading researchers who contribute from their respective scientific disciplines--and from their perspective at the cutting edge of the technology--this book * Combines a unified treatment and a critical evaluation of the field, presenting new ideas, methods, and instrumentation, with tips for developing new applications * Covers many new techniques in different scientific disciplines--from biology and chemistry to physics, engineering, and medicine * Emphasizes the multifunctional and the multidimensional uses of fluorescence spectroscopy and video microscopy, and shows how they can be applied in different combinations to obtain new information For biochemists, cell biologists, biophysicists, physiologists, neuroscientists, applied physicists, and materials scientists, as well as for scientists working in industrial, agricultural, medical, and pharmaceutical research--and for graduate students in these areas--this book is a novel resource of research opportunities, new ideas, and current practices in a dynamic and burgeoning new field.

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 book covers the fundamental principles of fluorescence and their application to fluorescence microscopy, and presents applications to immunofluorescence, in situ hybridization, and photomicrography. It provides troubleshooting guidance to guide the user through commonly encountered problems.

Fluorescence Microscopy of Living Cells in Culture, Part B

The detection and measurement of the dynamic interactions of proteins within the living cell are critical to our understanding of cell physiology and pathophysiology. With FRET microscopy and spectroscopy techniques, basic and clinical scientists can make such measurements at very high spatial and temporal resolution. But sources of background information about these tools are very limited, so this book fills an important gap. It covers both the basic concepts and theory behind the various FRET microscopy and spectroscopy techniques, and the practical aspects of using the techniques and analyzing the results. The critical tricks for obtaining a good FRET image and precisely quantitating the signals from living specimens at the nanomolecular level are explained. Valuable information about the preparation of biological samples used for FRET image analysis is also provided. The methods covered include different types of microscopy systems and detectors (wide-field, confocal, multi-photon) as well as specialized techniques such as photobleaching FRET, FLIM-FRET microscopy, spectral imaging FRET, single molecule FRET, and time and image correlation spectroscopy. Starting from the basics, the chapters guide readers through the choice of probes to be used for FRET experiments and the selection of the most suitable experimental approaches to address specific biological questions. Up-to-date, consistently organized, and well-illustrated, this unique book will be welcomed by all researchers who wish to use FRET microscopy and spectroscopy techniques.

The third edition of this established classic text reference builds upon the strengths of its very popular predecessors. Organized as a broadly useful textbook Principles of Fluorescence Spectroscopy, 3rd edition maintains its emphasis on basics, while updating the examples to include recent results from the scientific literature. The third edition includes new chapters on single molecule detection, fluorescence correlation spectroscopy, novel probes and radiative decay engineering. Includes a link to Springer Extras to download files reproducing all book artwork, for easy use in lecture slides. This is an essential volume for students, researchers, and industry professionals in biophysics, biochemistry, biotechnology, bioengineering, biology and medicine.