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.

Klappentext

This is the first book-length treatment of both the theoretical background to fluorescence correlation spectroscopy (FCS) and a variety of applications in various fields of science. The high spatial and temporal resolution of FCS has made it a powerful tool for the analysis of molecular interactions and kinetics, transport properties due to thermal motion, and flow. It contains an essential contribution from Nobel Prize winner M. Eigen, who is credited with inventing FCS.

Inhalt

1. Introduction.- References.- I FCS in the Analysis of Molecular Interactions.- 2 Fluorescence Correlation Spectroscopy of Flavins and Flavoproteins.- 2.1 Introduction.- 2.2 Materials and Methods.- 2.3 Results and Discussion.- 2.3.1 FCS on FMN and FAD.- 2.3.2 FCS on YFP and BFP.- 2.4 Conclusions.- References.- 3 Fluorescence Correlation Spectroscopy in Nucleic Acid Analysis.- 3.1 Introduction.- 3.2 Oligonucleotide-Target Interactions.- 3.3 DNA Analysis by "Going Micro".- 3.4 Incorporation of Dye Nucleotides into DNA.- 3.4.1 Low-Density Labeling.- 3.4.2 Nick Translation.- 3.4.3 Linear Primer Extension Reactions.- 3.4.4 High-Density Labeling.- 3.5 Exonuclease Degradation.- 3.6 Restriction Enzyme Cutting and DNA Sizing.- 3.7 Polymerase Chain Reaction.- 3.7.1 FCS Autocorrelation Analysis: New Detection Methods.- 3.7.2 FCS Cross-Correlation Analysis: A New Concept for PCR.- 3.8 Summary and Conclusions.- References.- 4 Strain-Dependent Fluorescence Correlation Spectroscopy: Proposing a New Measurement for Conformational Fluctuations of Biological Macromolecules.- 4.1 Introduction.- 4.2 Theory.- 4.3 A Simple Example.- 4.4 Discussion.- 4.4.1 SD-FCS.- 4.4.2 Comparison of SD-FCS with Conventional FCS.- 4.4.3 Applications and Feasibility.- 4.4 Summary.- References.- 5 Applications of FCS to Protein-Ligand Interactions: Comparison with Fluorescence Polarization.- 5.1 Fluorescence Polarization versus FCS.- 5.2 Experimental Methods.- 5.3 HIV Protease.- 5.4 Death Domain Interactions.- 5.5 Antibody-Small Ligand Interactions.- 5.6 Antibody-Large Ligand Interactions.- 5.7 Conclusions.- References.- II FCS at the Cellular Level.- 6 FCS-Analysis of Ligand-Receptor Interactions in Living Cells.- 6.1 Introduction.- 6.2 Materials and Methods.- 6.2.1 Chemicals.- 6.2.2 Cell Culture.- 6.2.3 Fluorescence Correlation Spectroscopy (FCS).- 6.3 Results.- 6.3.1 Background Signal.- 6.3.2 Binding of Rh-Ligands to the Cell Membranes.- 6.3.3 Presentation of Ligand-Receptor Complexes with Distribution of Diffusion Times.- 6.3.4 Saturation of Binding.- 6.3.5 Specificity and Kinetics of Binding.- 6.3.6 Measurement of the Association Rate Constant.- 6.3.7 Effect of Pertussis Toxin on the Ligand Binding.- 6.3.8 Measurement of IC50.- 6.4 Discussion.- 6.4.1 Demonstration of Specific Binding.- 6.4.2 Nature of Ligand-Receptor Interaction.- 6.4.3 Binding Kinetics.- 6.4.4 Different Ligand-Receptor Complexes and Binding Sites/Receptor Subtypes.- 6.4.5 Allosteric Nature of Signal Transduction and Receptor Aggregation.- 6.4.6 Problems, Limitations, and Precautions.- 6.5 Future Perspectives and Cross-Correlation.- References.- 7 Fluorescence Correlation Microscopy (FCM): Fluorescence Correlation Spectroscopy (FCS) in Cell Biology.- 7.1 Introduction.- 7.2 Theory of Cellular FCS.- 7.2.1 FCS in Multi-component Systems.- 7.2.2 Detection of Molecular Association Without Explicit Analysis of the Diffusion Constant D.- 7.2.3 Determination of N for Distributions of Molecules Carrying Different Numbers of Fluorophores per Molecule.- 7.2.4 Intracellular FCS - Approximation of Local Equilibria.- 7.2.5 FCS in Small Volumes - The Problem of Fluorophore Depletion.- 7.2.6 FCS-Derived Parameters in Cell Biology.- 7.3 Instrumental Requirements for Intracellular FCS.- 7.3.1 Design of the Fluorescence Correlation Microscope.- 7.4 Applications of Intracellular FCM.- 7.4.1 FCM in the Analysis of Receptor Diffusion - Measurement Protocols for Intracellular FCM.- 7.4.2 FCM in the Analysis of Metabolic Conversions.- 7.4.3 Comparison of Cytoplasmic and Nuclear GFP.- 7.4.4 FCM in Cellular High Throughput Screening.- 7.5 Limitations and Perspectives of Cellular FCM.- 7.5.1 FCM-Specific Problems in Intracellular Research.- 7.5.2 Perspectives in Cellular FCM.- 7.5.3 Comparison of FCM with Other Techniques.- References.- 8 FCS and Spatial Correlations on Biological Surfaces.- 8.1 The Problem.- 8.2 The Solution.- 8.3 The Experiment.- 8.3.1 Generating Images Using a Confocal Microscope.- 8.3.2 Correlation Calculations.- 8.3.3 Correlation Function Analysis.- 8.3.4 Extracting the Amplitude Information.- 8.3.5 Technical Issues.- 8.4 Interpretation of Correlation Function Amplitudes.- 8.4.1 Cluster Densities.- 8.4.2 Degree of Aggregation.- 8.4.3 Multiple Populations.- 8.4.4 Dynamics of Aggregation.- 8.4.5 Intermolecular Interactions and Colocalization.- 8.5 Applications to Cell Surfaces.- 8.5.1 Receptor Distributions.- 8.5.2 Interactions in Coated Pits.- 8.5.3 Virus Assembly and Fusion.- 8.5.4 Other Applications and Future Prospects.- 8.6 Conclusions.- References.- 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.- 9.1 Introduction.- 9.2 Real-Time Monitoring of Enzymatic Activity by Dual-Color FCS.- 9.3 RAPID FCS and CFCA for Screening Applications.- 9.4 Applications in Evolutionary Biotechnolog.- 9.5 Outlook.- References.- 10 Nanoparticle Immunoassays: A new Method for Use in Molecular Diagnostics and High Throughput Pharmaceutical Screening based on Fluorescence Correlation Spectroscopy.- 10.1 Introduction.- 10.2 Theory.- 10.2.1 Competitive NPIA.- 10.2.2 Sandwich NPIA.- 10.2.3 Autocorrelation Amplitudes.- 10.3 Material and Methods.- 10.3.1 Substances.- 10.3.2 Equipment.- 10.3.3 Reactions.- 10.3.4 Simulations and Data Fitting.- 10.4 Results.- 10.4.1 Simulations.- 10.4.2 Experiments.- 10.5 Discussion.- References.- 11 Protein Aggregation Associated with Alzheimer and Prion Diseases.- 11.1 Introduction.- 11.2 Prion-Protein Multimerization.- 11.2.1 Conformation and State of Aggregation.- 11.2.2 Analysis of Multimerization by FCS.- 11.2.3 Influence of Fluorescence Labeling on the Multimerization Reaction.- 11.2.4 Kinetics of Spontaneous Multimerization.- 11.2.5 Seeded Multimerization of PrP.- 11.2.6 Summary of PrP Conformational Transitions.- 11.3 Amyloid ß-Peptide Multimerization.- 11.3.1 Spontaneous Multimerization.- 11.3.2 Seeded Aggregation as a Diagnostic Tool.- 11.4 Synopsis.- References.- IV Environmental Analysis and Monitoring.- 12 Application of FCS to the Study of Environmental Systems.- 12.1 Introduction.- 12.2 Nature and Characteristics of Aquatic and Terrestrial Colloids and Biopolymers.- 12.2.1 Nature of the Major Aquatic and Terrestrial Colloids.- 12.2.2 Aggregation Processes and Aggregate Structure.- 12.2.3 Potential Advantages and Limitations of FCS for Environmental Applications.- 12.3 Development of FCS for its Application to the Study of Environmental Systems.- 12.3.1 Colloids With Sizes Comparable to the Bea…