The phenomenon known as fluorescence is now widely used in the chemical and life sciences largely due to the development of highly sophisticated fluorescent probe chemistries and the commercial availability of these probes as well as the development of novel microscopy approaches. Introduction to Fluorescence helps readers acquire a sound understanding of basic fluorescence theory and practice. It describes general principles in a straightforward way and uses examples from a variety of disciplines to demonstrate them.
In color throughout, the book takes readers through the history of important discoveries to the most current advances. It introduces the fundamentals of the fluorescence phenomenon and gives detailed examples of fluorescence applications in the molecular life sciences, including biochemistry, biophysics, clinical chemistry and diagnostics, pharmaceutical science, and cell and molecular biology. The author presents the basic theories underlying the applications and offers in-depth information on practical aspects.
Along with a list of references in each chapter, the text incorporates more than 250 figures that clearly illustrate the concepts and gives the chemical structures of the most widely used fluorescent molecules. In addition, the appendix provides a "Rogue’s Gallery" of the most common errors and pitfalls to avoid.
Introduction
What Is Fluorescence and How Is It Used?
A Nano-History of Fluorescence
General Concepts
And the Rest
Absorption of Light
Electromagnetic Radiation: Characterization
Photons
Relevant Wavelength Range
Absorption of Light by Molecules
Effect of Conjugation on Absorption
Effects of Molecular Environment on Absorption
Practical Considerations
Instrumentation
Light Sources
Optical Filters
Monochromators
Polarizers
Detectors
Miscellaneous Topics
Emission and Excitation Spectra
Emission Spectra
Excitation Spectra
Delayed Fluorescence
Advanced Scanning Methods
Polarization and Anisotropy
Basic Principles
Some Applications of Polarization/Anisotropy
Depolarization via FRET
Time-Resolved Fluorescence
Excited State Lifetimes
Instrumentation Details
Weber’s Algorithm
Time Domain versus Frequency Domain
Magic Angles
Anisotropy Decay/Dynamic Polarization
Dynamic Polarization
Phasors
Quantum Yields and Quenching
Determination of QYs
Kasha–Vavilov Rule
Quenching
Quenching and Membrane Systems
Förster Resonance Energy Transfer
Background
Basic Principles
How Do We Determine the Efficiency of Energy Transfer (E)?
The Orientation Factor
How Do We Determine κ2?
Homo-Transfer of Electronic Excitation Energy
Examples of FRET Applications
Advanced Treatments
Brief Overview of Fluorescence Microscopy
Introduction of Fluorophores into Living Cells
Fluorescence Microscopy Approaches
Specialized Techniques
Fluorophores
Where Do Fluorophores Come From?
How Does One Choose a Fluorophore?
Intrinsic Probes
Extrinsic Probes
Photostability
Labeling Proteins In Vitro
Membrane Probes
Nucleic Acid Probes and Nucleotide/Nucleoside
Analogs
Biosensors
Ion Probes
pH Probes
Molecular Beacons
Voltage-Sensitive Dyes
Miscellaneous Probes
Fluorescent Proteins
Intrinsic Protein Fluorescence
Spectra
Polarization
Lifetime
Electronic Energy Transfer in Proteins
Use of Site-Directed Mutagenesis
Tryptophan Analogs
Applications
Protein Unfolding/Refolding
Protein–Protein and Protein–DNA Interactions
Tyrosine Studies
Phasors and Protein Fluorescence
Appendix: Rogue’s Gallery of Fluorescence Artifacts and Errors
Index
Additional Reading appears at the end of each chapter.
Biography
David M. Jameson is a professor in the Department of Cell and Molecular Biology at the John A. Burns School of Medicine, University of Hawaii at Manoa, having previously served there as professor and chairman of the Department of Biochemistry and Biophysics. He earned his Ph.D. in biochemistry from the University of Illinois at Urbana-Champaign, where his graduate thesis advisor was Professor Gregorio Weber. Prior to his move to the University of Hawaii, he was a postdoctoral fellow at the University of Illinois at Urbana-Champaign and the CNRS, University of Paris-South and he was an assistant professor in the Department of Pharmacology at the University of Texas Southwestern Medical Center. Dr. Jameson is the co-organizer of the International Weber Symposia on Innovative Fluorescence Methodologies in Biochemistry and Medicine, which have been held every three years since 1986 (since 1995 in Hawaii). He serves on the editorial boards of The Scientific World Journal, Analytical Biochemistry, and Methods and Applications in Fluorescence. He is a member of the advisory board for the Laboratory for Fluorescence Dynamics (supported by the National Institutes of Health) at the University of California, Irvine. He was the recipient of the 2004 Gregorio Weber Award for Excellence in Fluorescence Theory/Application.
"An essential contribution to educating scientists in the principles of fluorescence. It will also be an important addition to the libraries of practitioners applying the principles of molecular fluorescence."
—Ken Jacobson, Kenan Distinguished Professor of Cell Biology and Physiology, University of North Carolina at Chapel Hill"An exquisite compendium of fluorescence and its applications in biochemistry enriched by a very exciting historical perspective. This book will become a standard text for graduate students and other scientists."
—Drs. Zygmunt (Karol) Gryczynski and Ignacy Gryczynski, University of North Texas Health Science Center"… truly a masterwork, combining clarity, precision, and good humor. The reader, novice or expert, will be pleased with the text and will not stop reading. It is a formidable account of the fluorescence field, which has impacted the life sciences so considerably in the last 60 years."
—Jerson L. Silva, M.D., Ph.D., Professor and Director, National Institute of Science and Technology for Structural Biology and Bioimaging, Universidade Federal do Rio de Janeiro"Dr. Jameson’s book fills a gap in the literature available for the student approaching the applications of fluorescence techniques as well as for the researcher practicing them daily in the laboratory setting. The subject is presented by the author in a clear didactic manner; the anecdotes and historical references make the book truly a delight to read."
—Dr. Beniamino Barbieri, President, ISS Inc."An excellent textbook! This comprehensive overview written at an introductory level covers fundamental aspects, principles of instrumentation, and practical applications of biological fluorescence, while providing many valuable tips. In essence, this book reflects the excellent didactic skills and vast experience of the author in research and teaching fluorescence. Highly recommendable for undergraduate teaching. Also excellent material for active researchers who are learning to apply fluorescence methods."
—Prof. Luis Bagatolli, Center for Biomembrane Physics, Department of Biochemistry and Molecular Biology, University of Southern Denmark"I am very impressed by the author’s lucid treatment. This book is a ‘must read’ for students and researchers."
—Prof. Amitabha Chattopadhyay, Ph.D., FRSC, J.C. Bose Fellow, Outstanding Scientist, Centre for Cellular & Molecular Biology, Hyderabad
