Lakowicz Principles of Fluorescence Spectroscopy

1983
ISBN: 978-1-4615-7658-7
Verlag: Springer US
Format: PDF
Kopierschutz: 1 - PDF Watermark

E-Book, Englisch, 496 Seiten, eBook

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.

Zielgruppe

Research

Autoren/Hrsg.

Lakowicz, Joseph R.

Inhaltsverzeichnis

1. Introduction to Fluorescence.- 1.1. Jablonski Diagram.- 1.2. Characteristics of Fluorescence Emission.- 1.3. Fluorescence Lifetimes and Quantum Yields.- 1.4. Fluorescence Anisotropy.- 1.5. Time Scale of Molecular Processes in Solution.- 1.6. Fluorophores.- 2. Instrumentation for Fluorescence Spectroscopy.- 2.1. Excitation and Emission Spectra.- 2.2. Light Sources.- 2.3. Monochromators.- 2.4. Optical Filters.- 2.5. Photomultiplier Tubes.- 2.6. Photon-Counting versus Analog Detection of Fluorescence.- 2.7. Corrected Fluorescence Spectra.- 2.8. Effects of Sample Geometry.- 2.9. Absorption of Light and Deviations from the Beer-Lambert Law.- 3. Measurement of Fluorescence Lifetimes.- 3.1. Pulse Lifetime Measurements.- 3.2. Phase and Modulation Measurements of Fluorescence Lifetimes.- 3.3. Pulse Lifetime Measurements and Instrumentation.- 3.4. Analysis of Time-Resolved Decays of Fluorescence Intensity.- 3.5. Time-Resolved Emission Spectra.- 3.6. Phase Shift and Demodulation Measurements of Fluorescence Lifetimes.- 3.7. Analysis of Phase and Modulation Data.- 3.8. Color Effects in Photomultiplier Tubes.- 3.9. Evaluation of the Performance of Lifetime Instruments.- Problems.- 4. Phase-Sensitive Detection of Fluorescence.- 4.1. Theory of Phase-Sensitive Detection of Fluorescence.- 4.2. Analysis of Heterogeneous Fluorescence by Phase-Sensitive Detection.- 4.3. Prospectus on the Resolution of Heterogeneous Fluorescence by PSDF.- Problems.- 5. Fluorescence Polarization.- 5.1. Definitions of Polarization and Anisotropy.- 5.2. Theory for Polarization in Dilute Vitrified Solution.- 5.3. Polarization Spectra of Fluorophores.- 5.4. Measurement of Fluorescence Anisotropics.- 5.5. Extrinsic Causes of Fluorescence Depolarization.- 5.6. Effects of Rotational Diffusion on Fluorescence Anisotropics; The Perrin Equation.- 5.7. Biochemical Applications of Anisotropy Measurements.- Problems.- 6. Time-Dependent Decays of Fluorescence Anisotropy.- 6.1. Theory of Time-Resolved Decays of Anisotropy.- 6.2. Biochemical Applications of Time-Resolved Anisotropy Measurements.- 6.3. Differential Polarized Phase Fluorometry.- 6.4. Lifetime-Resolved Measurements of Fluorescence Anisotropy.- Problems.- 7. Effects of Solvents on Fluorescence Emission Spectra.- 7.1. Stokes Shifts and Solvent Relaxation.- 7.2. General Solvent Effects on Fluorescence Spectra: The Lippert Equation.- 7.3. Derivation of the Lippert Equation.- 7.4. Specific Solvent Effects.- 7.5. Biochemical Applications of Solvent Effects.- Problems.- 8. Mechanisms and Dynamics of Solvent Relaxation.- 8.1. Effects of Solvent Relaxation on Steady State Emission Spectra.- 8.2. Theories of Time-Dependent Solvent Relaxation.- 8.3. Phase-Modulation Studies of Solvent Relaxation.- 8.4. Time-Resolved Measurements of Solvent Relaxation.- 8.5. Lifetime-Resolved Measurements of Solvent Relaxation.- 8.6. Analysis of Solvent Relaxation by Phase-Sensitive Detection of Fluorescence.- 8.7. Analysis of Spectral Relaxation in Model Membranes and Proteins.- Problems.- 9. Quenching of Fluorescence.- 9.1. Quenchers of Fluorescence.- 9.2. Theory of Collisional Quenching.- 9.3. Theory of Static Quenching.- 9.4. Combined Dynamic and Static Quenching.- 9.5. Examples of Static and Dynamic Quenching.- 9.6. Deviations from the Stern-Volmer Equation; Quenching Sphere of Action.- 9.7. Origin of the Smoluchowski Equation.- 9.8. Biochemical Applications of Quenching.- Problems.- 10. Energy Transfer.- 10.1. Theory of Energy Transfer for a Donor-Acceptor Pair.- 10.2. Distance Measurements by Energy Transfer.- 10.3. Association Reactions of Macromolecules Revealed by Energy Transfer.- 10.4. Static and Dynamic Mobility of Macromolecules Revealed by Time-Resolved Decays of Donor Fluorescence.- 10.5. Energy Transfer in the Rapid Diffusion Limit.- 10.6. Energy Transfer in Solution.- 10.7. Analysis of the Transfer Efficiency in the Presence of Nondipolar Quenching Mechanisms.- Problems.- 11. Protein Fluorescence.- 11.1. Spectral Properties of the Aromatic Amino Acids.- 11.2. General Characteristics of Protein Fluorescence.- 11.3. Factors Affecting the Emission Spectra of Proteins.- 11.4. Fluorescence Lifetimes of Proteins.- 11.5. Resolution of Individual Tryptophan Residues in Proteins from Time-Resolved Decays of Fluorescence.- 11.6. Dynamics of Proteins Revealed by Fluorescence Methods.- Problems.- 12. Spectral Characteristics of Systems Which Undergo a Reversible Two-State Reaction.- 12.1. Reversible Two-State Reaction.- 12.2. Time-Resolved Decays of Fluorescence for a Reversible Two- State Reaction.- 12.3. Analysis of Excited-State Reactions by Phase-Modulation Fluorometry.- 12.4. Model Calculations for Phase-Modulation Fluorometry.- 12.5. The Excited State Protonation of Acridine.- 12.6. Phase-Modulation Studies of Excited State Reactions.- Appendix: Answers to Problems.- References.

Über Autor(innen)

Dr. J.R. Lakowicz
is Professor of Biochemistry at the University of Maryland School of Medicine and Director of the Center for Fluorescence Spectroscopy. Dr. Lakowicz has published over 400 scientific articles, has edited numerous books, holds 16 issued patents, and is the sole author of the widely used text,
Principles ofFluorescence Spectroscopy
, also published by Kluwer Academic/Plenum Publishers, now in its Second Edition.