This is the third volume in the Reviews in Fluorescence series. To date, two volumes have been both published and well received by the scientific community. Several book reviews have also favorably described the series as an "excellent compilation of material which is well balanced from authors in both the US and Europe". Of particular mention we note the recent book review in JACS by Gary Baker, Los Alamos. In this 3rd volume we continue the tradition of publishing leading edge and timely articles from authors around the world. We hope you find this volume as useful as past volumes, which promises to be just as diverse with regard to content. Finally, in closing, we would like to thank Dr Kadir Asian for the typesetting of the entire volume and our counterparts at Springer, New York, for its timely publication. Professor Chris D. Geddes Professor Joseph R. Lakowicz August 20*^ 2005.

Over the last decade, fluorescence has become the dominant tool in biotechnology and medical imaging. These exciting advances have been underpinned by the advances in time-resolved techniques and instrumentation, probe design, chemical / biochemical sensing, coupled with our furthered knowledge in biology. Complementary volumes 9 and 10, Advanced Concepts of Fluorescence Sensing: Small Molecule Sensing and Advanced Concepts of Fluorescence Sensing: Macromolecular Sensing, aim to summarize the current state of the art in fluorescent sensing. For this reason, Drs. Geddes and Lakowicz have invited chapters, encompassing a broad range of fluorescence sensing techniques. Some chapters deal with small molecule sensors, such as for anions, cations, and CO2, while others summarize recent advances in protein-based and macromolecular sensors. The Editors have, however, not included DNA or RNA based sensing in this volume, as this were reviewed in Volume 7 and is to be the subject of a more detailed volume in the near future.

An essential reference for any laboratory working in the analytical fluorescence glucose sensing field. The increasing importance of these techniques is typified in one emerging area by developing non-invasive and continuous approaches for physiological glucose monitoring. This volume incorporates analytical fluorescence-based glucose sensing reviews, specialized enough to be attractive to professional researchers, yet appealing to a wider audience of scientists in related disciplines of fluorescence.

Fluorescence spectroscopy continues its advance to more sophisticated methods and applications. As one looks over the previous decades, its appears that the first practical instruments for time-resolved measurements appeared in the 1970's. The instrumentation and analysis methods for time-resolved fluorescence advanced rapidly throughout the 1980's. Since 1990 we have witnessed a rapid migration of the principles of time-resolved fluorescence to cell biology and clinical appli- tions. Most recently, we have seen the introduction of multi-photon excitation, pump-probe and stimulated emission methods for studies of biological mac- molecules and for cellular imaging. These advanced topics are the subject of the present volume. Two-photon excitation was first predicted by Maria Goppert-Mayer in 1931, but was not experimentally observed until 1961. Observation of two-photon excitation required the introduction of lasers which provided adequate photon density for multi-photon absorption. Since the early observations of two-photon excitation in the 1960s, multi-photon spectroscopy has been limited to somewhat exotic applications of chemical physics, where it is used to study the electronic symmetry of small molecules. Placing one's self back in 1980, it would be hard to imagine the use of multi-photon excitation in biophysics or cellular imaging.

During the past 15 years, there has been remarkable progress in the analysis and manipulation of DNA and its use in nanotechnology. DNA analysis is ubiquitous in molecular biology, medical diagnostics, and forensics. Much of the readout technology is based on fluorescence detection. This volume contains contributions from many experts in the field who present an overview of many aspects of DNA technology. These chapters provide an understanding of the underlying principles and technology, rather than an exhaustive review of the literature. Written in a clear straightforward style, this book is an excellent introduction for any scientist to the use of fluorescence in DNA analysis. DNA Technology is an essential reading for all academics, bench scientists, and industry professionals wishing to take advantage of the latest and greatest in this continuously emerging field. Key Features: *Comprehensive overview of the complexities of DNA analysis, *Covers topics of universal interest to a broad field of scientists, *Accessible utility in presenting state-of-the-art DNA technology, *Chapters authored by key figures in the field.

In this inaugural volume of a new series, experts in the field help biochemists, analytical chemists, spectroscopists, biophysicists, and other specialists keep up with the latest techniques and technologies available in fluorescence spectroscopy.

Fluorescence spectroscopy and its applications to the physical and life sciences have evolved rapidly during the past decade. The increased interest in fluorescence appears to be due to advances in time resolution, methods of data analysis and improved instrumentation. With these advances, it is now practical to perform time-resolved measurements with enough resolution to compare the results with the structural and dynamic features of mac- molecules, to probe the structures of proteins, membranes, and nucleic acids, and to acquire two-dimensional microscopic images of chemical or protein distributions in cell cultures. Advances in laser and detector technology have also resulted in renewed interest in fluorescence for clinical and analytical chemistry. Because of these numerous developments and the rapid appearance of new methods, it has become difficult to remain current on the science of fluorescence and its many applications. Consequently, I have asked the experts in particular areas of fluorescence to summarize their knowledge and the current state of the art. This has resulted in the initial three volumes of Topics in Fluorescence Spectroscopy, which is intended to be an ongoing series which summarizes, in one location, the vast literature on fluorescence spectroscopy. These first three volumes are designed to serve as an advanced text. These volumes describe the more recent techniques and technologies (Volume 1), the principles governing fluorescence and the experimental observables (Volume 2), and applications in biochemistry and biophysics (Volume 3).

Time-resolved fluorescence spectroscopy is widely used as a research tool in bioch- istry and biophysics. These uses of fluorescence have resulted in extensive knowledge of the structure and dynamics of biological macromolecules. This information has been gained by studies of phenomena that affect the excited state, such as the local environment, quenching processes, and energy transfer. Topics in Fluorescence Spectroscopy, Volume 4: Probe Design and Chemical Sensing reflects a new trend, which is the use of time-resolved fluorescence in analytical and clinical chemistry. These emerging applications of time-resolved fluorescence are the result of continued advances in laser detector and computer technology. For instance, pho- multiplier tubes (PMT) were previously bulky devices. Miniature PMTs are now available, and the performance of simpler detectors is continually improving. There is also considerable effort to develop fluorophores that can be excited with the red/ne- infrared (NIR) output of laser diodes. Using such probes, one can readily imagine small time-resolved fluorometers, even hand-held devices, being used fordoctor's office or home health care.

Fluorescence spectroscopy and its applications to the physical and life sciences have evolved rapidly during the past decade. The increased interest in fluorescence appears to be due to advances in time resolution, methods of data analysis, and improved instrumentation. With these advances, it is now practical to perform time-resolved measurements with enough resolution to compare the results with the structural and dynamic features of mac- molecules, to probe the structures of proteins, membranes, and nucleic acids, and to acquire two-dimensional microscopic images of chemical or protein distributions in cell cultures. Advances in laser and detector technology have also resulted in renewed interest in fluorescence for clinical and analytical chemistry. Because of these numerous developments and the rapid appearance of new methods, it has become difficult to remain current on the science of fluorescence and its many applications. Consequently, I have asked the experts in particular areas of fluorescence to summarize their knowledge and the current state of the art. This has resulted in the initial two volumes of Topics in Fluorescence Spectroscopy, which is intended to be an ongoing series which summarizes, in one location, the vast literature on fluorescence spectroscopy. The third volume will appear shortly. The first three volumes are designed to serve as an advanced text. These volumes describe the more recent techniques and technologies (Volume 1), the principles governing fluorescence and the experimental observables (Volume 2), and applications in biochemistry and biophysics (Volume 3).

Reviews in Fluorescence 2004, the first book of a new book series from Springer, is a collection of current trends and emerging hot topics in the field of Fluorescence.

This annual review series differs from Springer's current Topics in Fluorescence series in that it is more specialized and includes reviews of an individual's own work or scientific perspective. Reviews in Fluorescence will therefore complement the other fluorescence titles published by Springer, whilst feeding the requirement from the fluorescence community for annual informative updates and developments.

Key features:

- Reviews in Fluorescence will be citable, indexed, and available both in print and online.

- Reviews in Fluorescence will be published annually.

- Reviews in Fluorescence will comprise invited review articles that summarize the yearly progress in fluorescence.

- Alternate years will publish the Invited Papers from the Methods and Applications in Fluorescence conference series (MAFS).

Over the last decade, fluorescence has become the dominant tool in biotechnology and medical imaging. These exciting advances have been underpinned by the advances in time-resolved techniques and instrumentation, probe design, chemical / biochemical sensing, coupled with our furthered knowledge in biology.

Complementary volumes 9 & 10, Advanced Concepts of Fluorescence Sensing: Small Molecule Sensing and Advanced Concepts of Fluorescence Sensing: Macromolecular Sensing, aim to summarize the current state of the art in fluorescent sensing. For this reason, Drs. Geddes and Lakowicz have invited chapters, encompassing a broad range of fluorescence sensing techniques. Some chapters deal with small molecule sensors, such as for anions, cations, and CO2, while others summarize recent advances in protein-based and macromolecular sensors. The Editors have, however, not included DNA or RNA based sensing in this volume, as this were reviewed in Volume 7 and is to be the subject of a more detailed volume in the near future.

Last year we launched Volume 1 of the Reviews in Fluorescence series. The volume was well-received by the fluorescence community, with many e-mails and letters providing valuable feedback, we subsequently thank you all for your continued support. After the volume was published we were most pleased to learn that the volume is to be citable and indexed, appearing on the ISI database. Subsequently, as well as the series having an impact number in due course, individual chapters will appear on the database and be both citable and keyword searchable. We feel that this will be a powerful resource to both authors and readers, further disseminating leading-edge fluorescence based material. Our intention with this new series is to both disseminate and archive the most recent developments in both past and emerging fluorescence based disciplines. While all chapters are invited, we welcome and indeed encourage the fluorescence community to suggest areas of interest that they feel need to be covered by the series. In this new volume. Reviews in Fluorescence 2005, Volume 2, we have invited reviews in areas such as: Multi-dimensional Time-correlated Single Photon Counting; Fluorescence Correlation Spectroscopy; RNA folding; Lanthanide Probes and Fluorescent Biosensors to name but just a few. We hope you find this volume a useful resource and we look forward to receiving any suggestions you may have. Finally we would like to thank the authors for their timely articles, Caroleann Aitken for the fi-ont cover design, Kadir Asian for typesetting and Mary Rosenfeld for administrative support.

During recent years our enthusiasm for Radiative Decay Engineering (RDE) has continually increased. Many of the early predictions have been confirmed experimentally. We see numerous applications for RDE in biotechnology, clinical assays and analytical chemistry. While implementation of RDE is relatively simple, understanding the principles of RDE is difficult. The concepts are widely distributed in the optics and chemical physics literature, often described in terms difficult to understand by biophysical scientists.

RDE includes chapters from the experts who have studied metal particle optics and fluorophore-metal interactions. This collection describes the fundamental principles for the widespread use of radiative decay engineering in the biological sciences and nanotechnology.

The Journal of Fluorescencea (TM)s fifth Whoa (TM)s Who directory is to publish the names, contact details, specialty keywords, and a brief description of scientists employing fluorescence methodology and instrumentation in their working lives. In addition, the directory will provide company contact details with a brief list of fluorescence-related products. The directory will be edited by Chris D. Geddes and Joseph R. Lakowicz, editor and founding editor of the Journal of Fluorescence.

The Journal of Fluorescence's fourth Who's Who directory is to publish the names, contact details, specialty keywords, and a brief description of scientists employing fluorescence methodology and instrumentation in their working lives. In addition, the directory will provide company contact details with a brief list of fluorescence-related products. The directory will be edited by Chris D. Geddes and Joseph R. Lakowicz, editor and founding editor of the Journal of Fluorescence.

An essential reference for any laboratory working in the analytical fluorescence glucose sensing field. The increasing importance of these techniques is typified in one emerging area by developing non-invasive and continuous approaches for physiological glucose monitoring. This volume incorporates analytical fluorescence-based glucose sensing reviews, specialized enough to be attractive to professional researchers, yet appealing to a wider audience of scientists in related disciplines of fluorescence.

This is the third volume in the Reviews in Fluorescence series. To date, two volumes have been both published and well received by the scientific community. Several book reviews have also favorably described the series as an "excellent compilation of material which is well balanced from authors in both the US and Europe". Of particular mention we note the recent book review in JACS by Gary Baker, Los Alamos. In this 3rd volume we continue the tradition of publishing leading edge and timely articles from authors around the world. We hope you find this volume as useful as past volumes, which promises to be just as diverse with regard to content. Finally, in closing, we would like to thank Dr Kadir Asian for the typesetting of the entire volume and our counterparts at Springer, New York, for its timely publication. Professor Chris D. Geddes Professor Joseph R. Lakowicz August 20*^ 2005.

During the past 15 years, there has been remarkable progress in the analysis and manipulation of DNA and its use in nanotechnology. DNA analysis is ubiquitous in molecular biology, medical diagnostics, and forensics. Much of the readout technology is based on fluorescence detection. This volume contains contributions from many experts in the field who present an overview of many aspects of DNA technology. These chapters provide an understanding of the underlying principles and technology, rather than an exhaustive review of the literature. Written in a clear straightforward style, this book is an excellent introduction for any scientist to the use of fluorescence in DNA analysis.

DNA Technology is an essential reading for all academics, bench scientists, and industry professionals wishing to take advantage of the latest and greatest in this continuously emerging field.

Key Features:
*Comprehensive overview of the complexities of DNA analysis,
*Covers topics of universal interest to a broad field of scientists,
*Accessible utility in presenting state-of-the-art DNA technology,
*Chapters authored by key figures in the field.

The Journal of Fluorescence's first Who's Who directory is to publish the names, contact details, specialty keywords and a brief description of scientists employing fluorescence methodology and instrumentation in their working lives. In addition the directory will provide company contact details with a brief list of fluorescence related products. Nothing like this has been published before for the Fluorescence field.

Time-resolved fluorescence spectroscopy is widely used as a research tool in bioch- istry and biophysics. These uses of fluorescence have resulted in extensive knowledge of the structure and dynamics of biological macromolecules. This information has been gained by studies of phenomena that affect the excited state, such as the local environment, quenching processes, and energy transfer. Topics in Fluorescence Spectroscopy, Volume 4: Probe Design and Chemical Sensing reflects a new trend, which is the use of time-resolved fluorescence in analytical and clinical chemistry. These emerging applications of time-resolved fluorescence are the result of continued advances in laser detector and computer technology. For instance, pho- multiplier tubes (PMT) were previously bulky devices. Miniature PMTs are now available, and the performance of simpler detectors is continually improving. There is also considerable effort to develop fluorophores that can be excited with the red/ne- infrared (NIR) output of laser diodes. Using such probes, one can readily imagine small time-resolved fluorometers, even hand-held devices, being used fordoctor's office or home health care.

Fluorescence spectroscopy and its applications to the physical and life sciences have evolved rapidly during the past decade. The increased interest in fluorescence appears to be due to advances in time resolution, methods of data analysis and improved instrumentation. With these advances, it is now practical to perform time-resolved measurements with enough resolution to compare the results with the structural and dynamic features of mac- molecules, to probe the structures of proteins, membranes, and nucleic acids, and to acquire two-dimensional microscopic images of chemical or protein distributions in cell cultures. Advances in laser and detector technology have also resulted in renewed interest in fluorescence for clinical and analytical chemistry. Because of these numerous developments and the rapid appearance of new methods, it has become difficult to remain current on the science of fluorescence and its many applications. Consequently, I have asked the experts in particular areas of fluorescence to summarize their knowledge and the current state of the art. This has resulted in the initial three volumes of Topics in Fluorescence Spectroscopy, which is intended to be an ongoing series which summarizes, in one location, the vast literature on fluorescence spectroscopy. These first three volumes are designed to serve as an advanced text. These volumes describe the more recent techniques and technologies (Volume 1), the principles governing fluorescence and the experimental observables (Volume 2), and applications in biochemistry and biophysics (Volume 3).

Fluorescence spectroscopy and its applications to the physical and life sciences have evolved rapidly during the past decade. The increased interest in fluorescence appears to be due to advances in time resolution, methods of data analysis, and improved instrumentation. With these advances, it is now practical to perform time-resolved measurements with enough resolution to compare the results with the structural and dynamic features of mac- molecules, to probe the structures of proteins, membranes, and nucleic acids, and to acquire two-dimensional microscopic images of chemical or protein distributions in cell cultures. Advances in laser and detector technology have also resulted in renewed interest in fluorescence for clinical and analytical chemistry. Because of these numerous developments and the rapid appearance of new methods, it has become difficult to remain current on the science of fluorescence and its many applications. Consequently, I have asked the experts in particular areas of fluorescence to summarize their knowledge and the current state of the art. This has resulted in the initial two volumes of Topics in Fluorescence Spectroscopy, which is intended to be an ongoing series which summarizes, in one location, the vast literature on fluorescence spectroscopy. The third volume will appear shortly. The first three volumes are designed to serve as an advanced text. These volumes describe the more recent techniques and technologies (Volume 1), the principles governing fluorescence and the experimental observables (Volume 2), and applications in biochemistry and biophysics (Volume 3).

In this inaugural volume of a new series, experts in the field help biochemists, analytical chemists, spectroscopists, biophysicists, and other specialists keep up with the latest techniques and technologies available in fluorescence spectroscopy.

Reviews in Fluorescence 2004, the first book of a new book series from Springer, is a collection of current trends and emerging hot topics in the field of Fluorescence. This annual review series differs from Springer's current Topics in Fluorescence series in that it is more specialized and includes reviews of an individual's own work or scientific perspective. Reviews in Fluorescence will therefore complement the other fluorescence titles published by Springer, whilst feeding the requirement from the fluorescence community for annual informative updates and developments. Key features: - Reviews in Fluorescence will be citable, indexed, and available both in print and online. - Reviews in Fluorescence will be published annually. - Reviews in Fluorescence will comprise invited review articles that summarize the yearly progress in fluorescence. - Alternate years will publish the Invited Papers from the Methods and Applications in Fluorescence conference series (MAFS).

In the last few years, green fluorescent protein (GFP) has become one of the most widely used tools in molecular genetics. The fact that GFP can generate an internal highly visible fluorophore has made it a tremendously valuable marker or reporter gene which can be utilised in virtually every field of molecular cell genetics

One of the problems associated with this method is how to confirm that the gene of interest is being either expressed or activated in the target cells and to what extent this activation or expression occurs. The use of conventional reporter genes such as luciferase or lacZ require that the cells be processed and/or treated with substrates in order to monitor the reporter gene. The fact that GFP requires no additional substrates and can therefore be montiored non-invasively in living cells has been a revolutionary advance. In addition, a number of mutations in the wild type GFP protein, mean that it can also be used as a quantitative marker in living cells. GFP can also be used as a tag for studying localisation and movement of protein-GPF chimeras within cells in real time. There are also a growing number of GFP mutants with different spectral properties which allow, for example, simultaneous monitoring of proteins labelled with different GFPs or the use of fluorescent energy transfer techniques to measure protein-protein interations.