Fluorescence Correlation Spectroscopy

This book deals with an immportant subject in analytical chemistry and spectroscopy. It contains an essential contribution of the founder of this technique, the Nobel Prize winner M. Eigen.

First book on this important analytical technique

Klappentext
This book presents the theoretical background to fluorescence correlation spectroscopy (FCS) and a variety of applications in various fields of science. FCS is based on the detection of single molecules excited to fluorescence in diffraction limited confocal volume elements and the time correlation of stochastic events. It provides ultimate sensitivity in the analysis of molecular processes and has found numerous applications in physics, chemistry and particularly in biomolecular sciences. Its high spatial and temporal resolution has made FCS a powerful tool for the analysis of molecular interactions and kinetics, transport properties due to thermal motion and flow, as well as the physics of the excited state in solution as well as at the cellular level. Its application in high throughput drug screening is using all the potential of this prime analytical tool.

Zusammenfassung
Fluorescence correlation spectroscopy (FCS) was developed in order to char­ acterize the dynamics of molecular processes in systems in thermodynamic equilibrium. FCS determines transport and chemical reaction rates from mea­ surements of spontaneous microscopic thermally driven molecular concentra­ tion fluctuations. Since its inception, and particularly in recent years, techni­ cal and conceptual advances have extended the range of practical applicability and the information obtainable from FCS measurements. Improvements in microscopy, data acquisition, and data processing have greatly shortened the time required for FCS measurements. FCS can now be routinely applied to labile systems such as cells, and for the acquisition of large volumes of data as required for high-throughput screening. Cross correlation methods pro­ vide a powerful tool for characterizing interactions among different molecular species. Analysis of the amplitude of concentration fluctuations can provide a wealth of information about aggregation/polymerization process and the compositions of mixtures. Furthermore, FCS provides a bridge between conventional measurements of dynamic processes on a macroscopic concentration scale and the currently developing field of single molecule measurements. Both FCS and single mole­ cule approaches measure directly stochastic fluctuations in molecular pro­ perties, and so must be analyzed by statistical methods to yield conventional phenomenological parameters. As commonly practiced, FCS yields these phe­ nomenological parameters, e. g. , diffusion coefficients and chemical rate con­ stants, directly in terms of a fluorescence fluctuation autocorrelation func­ tion.

Inhalt

1. Introduction.- References.- I FCS in the Analysis of Molecular Interactions.- 2 Fluorescence Correlation Spectroscopy of Flavins and Flavoproteins.- 3 Fluorescence Correlation Spectroscopy in Nucleic Acid Analysis.- 4 Strain-Dependent Fluorescence Correlation Spectroscopy: Proposing a New Measurement for Conformational Fluctuations of Biological Macromolecules.- 5 Applications of FCS to Protein-Ligand Interactions: Comparison with Fluorescence Polarization.- II FCS at the Cellular Level.- 6 FCS-Analysis of Ligand-Receptor Interactions in Living Cells.- 7 Fluorescence Correlation Microscopy (FCM): Fluorescence Correlation Spectroscopy (FCS) in Cell Biology.- 8 FCS and Spatial Correlations on Biological Surfaces.- III Applications in Biotechnology, Drug Screening, and DiagnosticsPart 2 FCS at the Cellular Level.- 9 Dual-Color Confocal Fluorescence Spectroscopy and its Application in Biotechnology.- 10 Nanoparticle Immunoassays: A new Method for Use in Molecular Diagnostics and High Throughput Pharmaceutical Screening based on Fluorescence Correlation Spectroscopy.- 11 Protein Aggregation Associated with Alzheimer and Prion Diseases.- IV Environmental Analysis and Monitoring.- 12 Application of FCS to the Study of Environmental Systems.- 13 Photophysical Aspects of FCS Measurements.- V New Developments and Trends.- 14 Fluorescence Correlation Spectroscopy: Genesis, Evolution, Maturation and Prognosis.- 15 ConfoCor 2 The Second Generation of Fluorescence Correlation Microscopes.- 16 Antibunching and Rotational Diffusion in FCS.- 17 Cross-correlation analysis in FCS.- 18 Cross-correlated Flow Analysis in Microstructures.- 19 Introduction to the Theory of Fluorescence Intensity Distribution Analysis.- 20 Photon Counting Histogram Statistics.- 21 High Order Autocorrelationin Fluorescence Correlation Spectroscopy.- 22 FCS in Single Molecule Analysis.