Fluorescence Correlation Spectroscopy

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.