Time-Resolved Fluorescence Spectroscopy in Biochemistry and Biology

At the time that the editors conceived the idea of trying to organize the meeting on which the contents of this volume are based and which became, in March 1980, a NATO Advanced Study Institute, the techniques of time-resolved fluorescence spectroscopy, in both the nanosecond and sub-nanosecond time-domains, might reasonably have been said to be coming of age, both in their execution and in the analysis and interpretation of the results obtained. These techniques, then as now, comprised mainly a number of pulse methods using laser, flash-lamp or, most recently, synchrotron radiation. In addition, significant developments in the more classical phase approach had also rendered that method popular, utilizing either modulation of an otherwise continuous source or, again recently, the ultra-rapid pulse rate attainable with a synchrotron source. In general terms, time-resolved fluorescence studies are capable, under appropriate conditions, of supplying direct kinetic information on both photophysics and various aspects of molecular, macromolecular and supramolecular structure and dynamics. The nanosecond and sub-nanosecond time-scales directly probed render these techniques particularly appropriate in studying relaxation and fluctuation processes in macromolecules, particularly biopolymers (e. g. proteins, nucleic acids), in supramolecular assemblies such as cell membranes, and in a variety of relatively simpler model systems.

Inhalt

Old and New Developments in Fluorescence Spectroscopy.- Old and New Developments in Fluorescence Spectroscopy.- Technique.- Technique of Pulse Fluorometry.- Phase and Modulation Fluorometry.- Synchrotron Radiation as a Source to Study Time-Dependent Phenomena.- Remarks on a Novel Method for Fluorescence Lifetime Measurements.- Data Analysis.- The Pile-Up Problem in Pulse Fluorometry.- Least Squares Methods of Analysis.- Least Squares Methods of Analysis.- A Critical Appraisal of Analytical Methods.- Estimators of Exponential Decay - A Postscript.- Beyond Chi-Square: Evaluation of Parametric Models Used in the Analysis of Data from Fluorescence Decay Experiments.- Fluorescence Decay Analysis by the Method of Moments.- Remarks on the Method of Moments for Fluorescence Decay Analysis.- Systems Analysis Approaches.- Fourier Transform Analysis.- Analysis of Pulse Fluorometry Data by Laplace Transforms.- Modulating Functions - A Deconvolution Method.- An Analysis of Errors in the Phase-Plane Method of Deconvoluting Luminescence Lifetimes.- Excited-State Kinetics.- Kinetics of Fluorescence Decay: An Overview.- Excited-State Proton Transfer.- Kinetic Aspects of Exciplex Photophysics.- The Influence of Diffusion on Fluorescence Quenching.- Solvent Relaxation.- Long-Range Nonradiative Transfer of Electronic Excitation Energy.- Energy Migration in Disordered Systems.- Convolution Kinetics.- Fluorescence Depolarization.- Measurement of Brownian Correlation Times by Pulse Fluorometry.- Fluorescence Anisotropy Decay and Brownian Rotational Motion: Theory and Application in Biological Systems.- Energy Migration and Fluorescence Depolarization: Structural Studies of Ethidium Bromide-Nucleic Acid Complexes.- Theory and Application of Differential Polarized Phase Fluorometry.- Membrane Structure and Dynamics by Fluorescence Probe Depolarization Kinetics.- Selected Applications.- Comments on the Tryptophan Lifetime Puzzle.- The Excited-State Deprotonation of the Indole N-H of Tryptophan.- A Preliminary Application of Phase/Modulation Fluorometry to the Tryptophan Problem.- The Photophysics of Tryptophan.- The Fluorescence Decays of Tryptophan in Solution at Neutral pH and in Horse Liver Alcohol Dehydrogenase.- Time-resolved Spectroscopy of Tryptophyl Fluorescence in Proteins: Application to Yeast 3-Phosphoglycerate Kinase.- Intrinsic Fluorescence of Proteins.- Fluorescence in Aqueous Micellar Solutions.- Energy Migration in Photosynthesis.- Time-Resolved Fluorescence and Photosynthesis.- Epilogue.- Epilogue.