Fluorescence Correlation Spectroscopy

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Beschreibung

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.

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.

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.- 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 Beam Width.- 12.3.2 Polydisperse Systems.- 12.4 Example: Determination of the Diffusion Coefficients of Humic Substances as a Function of Solution Conditions.- 12.4.1 Factors Distinguishing Humic Substances From Model Compounds.- 12.4.2 The Role of Solution Conditions (pH, Ionic Strength, Concentration) on the Diffusion Coefficients of Humic Substances.- 12.5 Conclusions and Future Perspectives.- References.- 13 Photophysical Aspects of FCS Measurements.- 13.1 Introduction.- 13.2 Photophysics in the Fast Time Range.- 13.2.1 Triplet State Formation.- 13.2.2 Charge Transfer Reactions.- 13.2.3 Photo-Induced Isomerization.- 13.2.4 Effects of Non-Uniform Excitation.- 13.3 Photophysics in the Slow Time Range-Photodegradation.- 13.4 Strategies to Improve Photophysical Conditions.- 13.5 Concluding Remarks.- References.- V New Developments and Trends.- 14 Fluorescence Correlation Spectroscopy: Genesis, Evolution, Maturation and Prognosis.- 14.1 Introduction.- 14.2 Genesis.- 14.3 Evolution.- 14.4 Maturation of FCS at Cornell.- 14.4.1 Green Fluorescent Proteins in FCS.- 14.4.2 Molecular Diffusion in Lipid Membranes of Giant Unilamellar Vesicles.- 14.4.3 Two-Photon Molecular Excitation (2PE) for FCS.- 14.4.4 FCS in Cells and Tissues.- 14.5 Prognosis for FCS.- References.- 15 ConfoCor 2 The Second Generation of Fluorescence Correlation Microscopes.- 15.1 Introduction.- 15.2 Instrumental Setup.- 15.2.1 Laser Module.- 15.2.2 Detection Unit.- 15.2.3 Detection Efficiency Profile.- 15.2.4 Detection of Fuorescence Correlation Signals.- 15.2.5 FCS Data Analysis.- 15.3 Autocorrelation Measurements.- 15.4 Cross Correlation Measurements.- 15.5 Summary.- References.- 16 Antibunching and Rotational Diffusion in FCS.- 16.1 Introduction.- 16.2 Antibunching.- 16.3 Rotational Diffusion.- 16.4 Discussion.- References.- 17 Cross-correlation analysis in FCS.- 17.1 Introduction.- 17.2 Theory.- 17.2.1 Fluctuation Correlations.- 17.2.2 The Effective Measurement Volume in FCS.- 17.2.3 Autocorrelation and Cross-correlation Functions for Pure Diffusion.- 17.2.4 Detector Cross-Talk.- 17.2.5 Not Completely Overlapping Detection Volumes.- 17.2.6 Cross-correlation of Internal Fluctuations.- 17.3 Experimental Realization.- 17.4 Applications.- 17.4.1 Slow Association Reactions: Comparison Between Autocorrelation and Cross-correlation.- 17.4.2 Cross-correlation Applications in Various Biochemical Systems.- 17.4.3 Outlook.- References.- 18 Cross-correlated Flow Analysis in Microstructures.- 18.1 Introduction.- 18.2 The Experimental Setup.- 18.3 Theory.- 18.3.1 Pseudo-Autocorrelation.- 18.4 Experimental Procedures.- 18.4.1 Optimizing the Setup.- 18.4.2 Flow Measurements.- 18.5 Applications.- 18.5.1 Continuous Flow Kinetics.- 18.5.2 Rapid DNA Sequencing.- 18.6 Conclusion.- References.- 19 Introduction to the Theory of Fluorescence Intensity Distribution Analysis.- 19.1 Introduction.- 19.2 Photon Count Number Distribution Corresponding to a Rectangular Sample Profile.- 19.3 Photon Count Number Distribution Corresponding to an Arbitrary Sample Profile: The Convolution Technique.- 19.4 Photon Count Number Distribution Corresponding to an Arbitrary Sample Profile: The Technique of the Generating Function.- 19.5 Sample Profile Models.- 19.6 Distribution of the Specific Brightness Within a Species.- 19.7 Weighting in FIDA.- 19.8 Data Simulation Algorithms.- 19.9 Statistical Errors of Estimated Parameters.- References.- 20 Photon Counting Histogram Statistics.- 20.1 Introduction.- 20.2 Theory.- 20.3 PCH and the Theory of Photon Detection.- 20.3.1 PCH of a Single Particle.- 20.3.2 PCH of Multiple Particles.- 20.3.3 PCH of Particles with Number Fluctuations.- 20.3.4 PCH of Multiple Species.- 20.3.5 PCH for Different PSFs.- 20.3.6 Describing PCH with the Moment Generating Function.- 20.3.7 Two-fold PCH Statistics.- 20.4 Data Analysis.- 20.5 Single Species PCH.- 20.5.1 Influence of the Particle Concentration.- 20.5.2 Influence of Molecular Brightness.- 20.5.3 Sensitivity of PCH Algorithm.- 20.6 PCH for Multiple Species.- 20.6.1 Resolvability of Two Species.- 20.6.2 Experimental Results.- 20.7 Conclusions.- References.- 21 High Order Autocorrelation in Fluorescence Correlation Spectroscopy.- 21.1 Introduction.- 21.2 Temporal High Order FCS.- 21.2.1 Definitions.- 21.2.2 First Order Fluorescence Fluctuation Autocorrelation.- 21.2.3 High Order Fluorescence Fluctuation Autocorrelation.- 21.2.4 Multicomponent Analysis.- 21.2.5 Experimental Considerations.- 21.2.6 Experimental Applications.- 21.3 Spatial High Order FCS.- 21.3.1 Overview.- 21.3.2 Spatial Fluorescence Fluctuation Autocorrelation Functions.- 21.3.3 Autocorrelation Function Magnitudes and Decay Shapes.- 21.3.4 Experimental Considerations.- 21.3.5 Experimental Application.- 21.4 Discussion.- References.- 22 FCS in Single Molecule Analysis.- 22.1 Introduction.- 22.2 Single Molecule Detection in Solution and Correlation Functions.- 22.3 Confocal Single Molecule Imaging.- 22.4 Conformatial Transitions in Single DNA Molecules.- 22.5 Single Molecule Traces.- 22.6 Homogeneous and Heterogeneous Behavior.- 22.7 Time Resolution of Single Molecule Behaviour.- 22.8 Kinetic Analysis, Death Numbers, and Survival Times.- 22.9 The Fluctuating Enzyme.- 22.10 Evidence for Multiple Conformational Transition and Catalysis.- 22.11 Higher Order Correlations and Non-Markovian Behavior.- 22.12 Conclusions.- References.

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Produktinformationen

Titel
Fluorescence Correlation Spectroscopy
Untertitel
Theory and Applications
Editor
EAN
9783642640186
ISBN
3642640184
Format
Kartonierter Einband
Herausgeber
Springer Berlin Heidelberg
Anzahl Seiten
512
Gewicht
768g
Größe
H235mm x B155mm x T27mm
Jahr
2011
Untertitel
Englisch
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