R . Rigler E.S. Elson

FluorescenceCorrelatio nSpectroscopyTheory and Applications

List of Contributors XVII

1 IntroductionE. Elson 1

References 5

Part I FCS in the Analysis of Molecular Interactions

2 Fluorescence Correlation Spectroscopyof Flavins and Flavoprotein sAntonio J .W.G. Visser, Petra A.W. van den Berg, Mark A . Hink, andValentin N. Petushkov 9

2 .1

Introduction 92 .2 Materials and Methods 1 1

2 .3

Results and Discussion 1 22 .3 .1 FCS on FMN and FAD 1 22 .3 .2 FCS on YFP and BFP 1 8

2 .4

Conclusions 22References 23

3 Fluorescence Correlation Spectroscopyin Nucleic Acid AnalysisZeno Földes-Papp and Masataka Kinjo 25

3 .1

Introduction 25

3 .2

Oligonucleotide-Target Interactions 2 73 .3 DNA Analysis by "Going Micro" 3 1

3 .4 Incorporation of Dye Nucleotides into DNA 333 .4 .1

Low-Density Labeling 33

3 .4 .2

Nick Translation 36

3 .4 .3

Linear Primer Extension Reactions 3 73 .4 .4

High-Density Labeling 39

3 .5 Exonuclease Degradation 4 13 .6 Restriction Enzyme Cutting and DNA Sizing 43

3 .7 Polyrnerase Chain Reaction 46

3 .7 .1

FCS Autocorrelation Analysis : New Detection Methods 4 7

3 .7 .2

FCS Cross-Correlation Analysis : A New Concept for PCR 5 33 .8 Summary and Conclusions 5 9

References 6 0

4 Strain-Dependent Fluorescence Correlation Spectroscopy:

Proposing a New Measurement for Conformationa lFluctuations of Biological Macromolecule sHong Qian and Elliot L . Elson 6 5

4 .1

Introduction 6 54.2 Theory 6 74.3 A Simple Example 7 04 .4

Discussion 764 .4 .1

SD-FCS 764 .4 .2

Comparison of SD-FCS with Conventional FCS 804 .4 .3

Applications and Feasibility 8 14.5 Summary 82References 8 2

5 Applications of FCS to Protein-Ligand Interactions :Comparison with Fluorescence PolarizationEdmund Matayoshi and Kerry Swift 84

5 .1

Fluorescence Polarization versus FCS 845.2 Experimental Methods 875.3 HIV Protease 885.4 Death Domain Interactions 905.5 Antibody-Small Ligand Interactions 935.6 Antibody-Large Ligand Interactions 955.7 Conclusions 96References 97

Part II FCS at the Cellular Level

6 FCS-Analysis of Ligand-Receptor Interactionsin Living Cell sAladdin Pramanik and Rudolf Rigler 10 1

6 .1

Introduction 10 16.2 Materials and Methods 10 1

6 .2 .1

Chemicals 10 16 .2 .2

Cell Culture 10 26 .2 .3

Fluorescence Correlation Spectroscopy (FCS) 10 26 .3

Results 10 86 .3 .1

Background Signal 108

6 .3 .2

Binding of Rh-Ligands to the Cell Membranes 10 86 .3 .3

Presentation of Ligand-Receptor Complexeswith Distribution of Diffusion Times 11 3

6 .3 .4

Saturation of Binding 11 56 .3 .5

Specificity and Kinetics of Binding 11 76 .3 .6

Measurement of the Association Rate Constant 12 06 .3 .7

Effect of Pertussis Toxin on the Ligand Binding 12 16 .3 .8

Measurement of IC 50 12 16 .4

Discussion 12 26 .4 .1

Demonstration of Specific Binding 12 26 .4 .2

Nature of Ligand-Receptor Interaction 12 36 .4 .3

Binding Kinetics 12 46 .4 .4

Different Ligand-Receptor Complexe sand Binding Sites/Receptor Subtypes 12 4

6 .4 .5

Allosteric Nature of Signal Transductionand Receptor Aggregation 12 5

6 .4 .6

Problems, Limitations, and Precautions 12 76 .5 Future Perspectives and Cross-Correlation 12 8References 12 9

7 Fluorescence Correlation Microscopy (FCM) :Fluorescence Correlation Spectroscopy (FCS) in Cell Biolog yRoland Brock and Thomas M . Jovin 13 2

7 .1

Introduction 13 27 .2 Theory of Cellular FCS 13 3

7 .2 .1

FCS in Multi-component Systems 13 37 .2 .2

Detection of Molecular Association Without Explici tAnalysis of the Diffusion Constant D 13 4

7 .2 .3

Determination of N for Distributionsof Molecules Carrying Different Number sof Fluorophores per Molecule 13 5

7 .2 .4

Intracellular FCS -- Approximation of Local Equilibria 13 67 .2 .5

FCS in Small Volumes -The Problem of Fluorophore Depletion 13 7

7 .2 .6

FCS-Derived Parameters in Cell Biology 13 77.3 Instrumental Requirements for Intracellular FCS 13 8

7 .3 .1

Design of the Fluorescence Correlation Microscope 1387 .4 Applications of Intracellular FCM 142

7 .4.1

FCM in the Analysis of Receptor Diffusion -Measurement Protocols for Intracellular FCM 14 2

7 .4.2

FCM in the Analysis of Metabolic Conversions 1487 .4.3

Comparison of Cytoplasmic and Nuclear GFP 15 17 .4 .4

FCM in Cellular High Throughput Screening 1527 .5 Limitations and Perspectives of Cellular FCM 153

7 .5 .1

FCM-Specific Problems in Intracellular Research 153

7 .5 .2

Perspectives in Cellular FCM 15 57 .5 .3

Comparison of FCM with Other Techniques 15 7References 15 9

8 FCS and Spatial Correlations on Biological Surface sNils O. Petersen 16 2

8.1 The Problem 16 28 .2

The Solution 1638 .3 The Experiment 16 5

8 .3 .1

Generating Images Using a Confocal Microscope 1658 .3 .2

Correlation Calculations 16 58 .3 .3

Correlation Function Analysis 16 68 .3 .4

Extracting the Amplitude Information 16 78 .3 .5

Technical Issues 16 98 .4 Interpretation of Correlation Function Amplitudes 17 0

8 .4 .1

Cluster Densities 17 08 .4 .2

Degree of Aggregation 17 18 .4 .3

Multiple Populations 17 28 .4 .4

Dynamics of Aggregation 17 38 .4 .5

Intermolecular Interactions and Colocalization 17 38 .5

Applications to Cell Surfaces 1748 .5 .1

Receptor Distributions 17 58 .5 .2

Interactions in Coated Pits 1788 .5 .3

Virus Assembly and Fusion 1808.5 .4

Other Applications and Future Prospects 18 18 .6

Conclusions 18 1References 18 3

Part III Applications in Biotechnology,Drug Screening, and Diagnostic s

9 Dual-Color Confocal Fluorescence Spectroscopyand its Application in BiotechnologyAndre Koltermann*, Ulrich Kettling, Jens Stephan, Thorsten Winkler ,and Manfred Eigen 18 7

9 .1

Introduction 18 79.2 Real-Time Monitoring of Enzymatic Activity

by Dual-Color FCS 1899.3 RAPID FCS and CFCA for Screening Applications 193

9.4 Applications in Evolutionary Biotechnology 199

9.5

Outlook 20 1

References 202

10 Nanoparticle Immunoassays :A new Method for Use in Molecular Diagnostic sand High Throughput Pharmaceutical Screening based o nFluorescence Correlation SpectroscopyF.J . Meyer-Alines 204

10 .1 Introduction 20 410 .2 Theory 20 6

10 .2 .1 Competitive NPIA 20 610 .2 .2 Sandwich NPIA 20 710 .2 .3 Autocorrelation Amplitudes 20 9

10 .3 Material and Methods 21 010 .3 .1 Substances 21 010 .3.2 Equipment 21 110 .3 .3 Reactions 21 110 .3 .4 Simulations and Data Fitting 21 2

10 .4 Results 21 210 .4 .1

Simulations 21 210 .4.2 Experiments 21 3

10 .5 Discussion 22 1References 22 3

11 Protein Aggregation Associatedwith Alzheimer and Prion Disease sDetlev Riesner 22 5

11 .1 Introduction 22 511 .2 Prion-Protein Multimerization 22 6

11 .2 .1 Conformation and State of Aggregation 22 611 .2 .2 Analysis of Multimerization by FCS 22 911 .2 .3 Influence of Fluorescence Labeling

on the Multimerization Reaction 23 111 .2 .4 Kinetics of Spontaneous Multimerization 23 311 .2 .5 Seeded Multimerization of PrP 23 311 .2 .6 Summary of PrP Conformational Transitions 23 6

11 .3 Amyloid ß-Peptide Multimerization 23 711 .3 .1 Spontaneous Multimerization 23 711 .3 .2 Seeded Aggregation as a Diagnostic Tool 24 0

11 .4 Synopsis 24 4References 245

Part IV Environmental Analysis and Monitorin g

12 Application of FCS to the Studyof Environmental System sKonstantin Starchev, Kevin J . Wilkinson, and Jacques Bufile 25 1

12 .1 Introduction 25 112 .2 Nature and Characteristics of Aquatic

and Terrestrial Colloids and Biopolymers 25 212 .2 .1 Nature of the Major Aquatic and Terrestrial Colloids 25 212 .2 .2 Aggregation Processes and Aggregate Structure 25 712 .2 .3 Potential Advantages and Limitations of FC S

for Environmental Applications 25 912 .3 Development of FCS for its Applicatio n

to the Study of Environmental Systems 26 012 .3 .1 Colloids With Sizes Comparable to the Beam Width 26 012 .3.2 Polydisperse Systems 26 2

12 .4 Example: Determination of the Diffusion Coefficients of Humi cSubstances as a Function of Solution Conditions 26 612 .4 .1 Factors Distinguishing Humic Substances

From Model Compounds 26812 .4.2 The Role of Solution Conditions (pH, Ionic Strength ,

Concentration) on the Diffusion Coefficient sof Humic Substances 27 1

12 .5 Conclusions and Future Perspectives 273References 274

13 Photophysical Aspects of FCS Measurement sJerker Widengren 276

13 .1 Introduction 27613 .2 Photophysics in the Fast Time Range 278

13 .2.1 Triplet State Formation 28 113 .2 .2 Charge Transfer Reactions 28713 .2.3 Photo-Induced Isomerization 29013 .2 .4 Effects of Non-Uniform Excitation 293

13 .3 Photophysics in the Slow TimeRange-Photodegradation 294

13 .4 Strategies to Improve Photophysical Conditions 29613 .5 Concluding Remarks 299References 300

Part V New Developments and Trends

14 Fluorescence Correlation Spectroscopy :Genesis, Evolution, Maturation and PrognosisWatt W . Webb 30 5

14 .1 Introduction 30 514 .2 Genesis 30 514 .3 Evolution 31 014.4 Maturation of FCS at Cornell 31 4

14 .4 .1 Green Fluorescent Proteins in FCS 31 514 .4 .2 Molecular Diffusion iii Lipid Membrane s

of Giant Unilamellar Vesicles 31 914 .4 .3 Two-Photon Molecular Excitation (2PE) for FCS 32 114 .4 .4 FCS in Cells and Tissues 32 3

14 .5 Prognosis for FCS 32 6References 32 8

15 ConfoCor 2 The Second Generatio nof Fluorescence Correlation Microscope sTilo Jankowski . Reinhard Janka 33 1

15 .1 Introduction 33 115 .2 Instrumental Setup 33 1

15 .2 .1 Laser Module 33 215 .2 .2 Detection Unit 33 315 .2 .3 Detection Efficiency Profile 33 515 .2 .4 Detection of Fuorescence Correlation Signals 33 715 .2 .5 FCS Data Analysis 33 8

15 .3 Autocorrelation Measurements 33 915 .4 Cross Correlation Measurements 34 215 .5 Summary 34 5References 34 5

16 Antibunching and Rotational Diffusion in FC SUlo Mets 34 6

16 .1 Introduction 34 616 .2 Antibunching 34616 .3 Rotational Diffusion 35 416 .4 Discussion 35 8References 359

17 Cross-correlation analysis in FC S

Petra Schwille 36 0

17 .1 Introduction 36 0

17.2 Theory 36 3

17 .2 .1 Fluctuation Correlations 36 3

17 .2 .2 The Effective Measurement Volume in FCS 364

17 .2 .3 Autocorrelation and Cross-correlation Functions

for Pure Diffusion 36 6

17 .2 .4 Detector Cross-Talk 368

17 .2 .5 Not Completely Overlapping Detection Volumes 369

17 .2 .6 Cross-correlation of Internal Fluctuations 37017 .3 Experimental Realization 37 1

17.4 Applications

37317 .4 .1 Slow Association Reactions : Comparison

Between Autocorrelation and Cross-correlation 37317 .4 .2 Cross-correlation Applications

in Various Biochemical Systems 37 517 .4.3 Outlook 37 7

References 37 7

18 Cross-correlated Flow Analysis in Microstructure sMichael Brinkmeier 37 9

18 .1 Introduction 37 918 .2 The Experimental Setup 38 018.3 Theory 38 2

18 .3 .1 Pseudo-Autocorrelation 38 618 .4 Experimental Procedures 387

18 .4 .1 Optimizing the Setup 38718 .4 .2 Flow Measurements 387

18 .5 Applications 39018 .5 .1 Continuous Flow Kinetics 39 118 .5 .2 Rapid DNA Sequencing 393

18 .6 Conclusion 394References 39 5

19 Introduction to the Theor yof Fluorescence Intensity Distribution Analysi sPeet Kask and Kaupo Palo 39 6

19 .1 Introduction 39 619 .2 Photon Count Number Distribution

Corresponding to a Rectangular Sample Profile 39 819 .3 Photon Count Number Distribution

Corresponding to an Arbitrary Sample Profile :The Convolution Technique 398

19 .4 Photon Count Number DistributionCorresponding to an Arbitrary Sample Profile :The Technique of the Generating Function 399

19 .5 Sample Profile Models 40019 .6 Distribution of the Specific Brightness Within a Species 40 119 .7 Weighting in FIDA 40 219 .8 Data Simulation Algorithms 40319.9 Statistical Errors of Estimated Parameters 404References 409

20 Photon Counting Histogram StatisticsJoachim D . Müller, Yan Chen and Enrico Grafton 410

20 .1 Introduction 41 020 .2 Theory 41 120 .3 PCH and the Theory of Photon Detection 41 1

20 .3 .1 PCH of a Single Particle 41 420 .3 .2 PCH of Multiple Particles 41 420 .3 .3 PCH of Particles with Number Fluctuations 41 520 .3 .4 PCH of Multiple Species 41 620 .3 .5 PCH for Different PSFs 41 620 .3 .6 Describing PCH with the Moment Generating Function 41820 .3 .7 Two-fold PCH Statistics 42 0

20 .4 Data Analysis 42 120 .5 Single Species PCH 42 1

20 .5 .1 Influence of the Particle Concentration 42120 .5 .2 Influence of Molecular Brightness 42 320 .5 .3 Sensitivity of PCH Algorithm 42 5

20 .6 PCH for Multiple Species 42 820 .6 .1 Resolvability of Two Species 42 820 .6 .2 Experimental Results 43 0

20 .7 Conclusions 43 3References 43 5

21 High Order Autocorrelationin Fluorescence Correlation SpectroscopyNancy L . Thompson . Jennifer L . Mitchell 43 8

21 .1 Introduction 43 821 .2 Temporal High Order FCS 43 9

21 .2 .1 Definitions 43 921 .2 .2 First Order Fluorescence Fluctuation Autocorrelation 44 021 .2 .3 High Order Fluorescence Fluctuation Autocorrelation 44 221 .2 .4 Multicomponent Analysis 44 421 .2 .5 Experimental Considerations 44 721 .2 .6 Experimental Applications 449

21 .3 Spatial High Order FCS 45 121 .3 .1 Overview 45 1

21 .3 .2 Spatial Fluorescence Fluctuation Autocorrelatio nFunctions 45 2

21 .3 .3 Autocorrelation Function Magnitudes and Decay Shapes 45 2

21 .3 .4 Experimental Considerations 45321 .3 .5 Experimental Application 454

21 .4 Discussion 455References 456

22 FCS in Single Molecule AnalysisR. Rigler, S . Wennmalm, and L . Edman 459

22 .1 Introduction 45922 .2 Single Molecule Detection in Solution

and Correlation Functions 45922 .3 Confocal Single Molecule Imaging 46222 .4 Conformatial Transitions

in Single DNA Molecules 46322 .5 Single Molecule Traces 46422 .6 Homogeneous and Heterogeneous Behavior 46522 .7 Time Resolution of Single Molecule Behaviour 46522 .8 Kinetic Analysis, Death Numbers ,

and Survival Times 46722 .9 The Fluctuating Enzyme 46822 .10 Evidence for Multiple Conformational Transitio n

and Catalysis 46822 .11 Higher Order Correlation s

and Non-Markovian Behavior 47422 .12 Conclusions 474References 475

Subject Index 477