Fluorescence Correlation Spectroscopy: Theory and Applications

by Rigler, Rudolf; Elson, E. S.

• ISBN13: 9783540674337
• ISBN10: 3540674330
• Format: Hardcover
• Copyright: 2001-05-01
• Publisher: Springer Verlag

Summary

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.

Table of Contents

List of Contributors	xvii
Introduction	1	(8)
E. Elson
References	5	(4)
Part I FCS in the Analysis of Molecular Interactions
Fluorescence Correlation Spectroscopy of Flavins and Flavoproteins	9	(16)
Antonie J.W.G. Visser
Petra A.W. van den Berg
Mark A. Hink
Valentin N. Petushkov
Introduction	9	(2)
Materials and Methods	11	(1)
Results and Discussion	12	(10)
FCS on FMN and FAD	12	(6)
FCS on YFP and BFP	18	(4)
Conclusions	22	(3)
References	23	(2)
Fluorescence Correlation Spectroscopy in Nucleic Acid Analysis	25	(40)
Zeno Foldes-Papp
Masataka Kinjo
Introduction	25	(2)
Oligonucleotide-Target Interactions	27	(4)
DNA Analysis by ``Going Micro''	31	(2)
Incorporation of Dye Nucleotides into DNA	33	(8)
Low-Density Labeling	33	(3)
Nick Translation	36	(1)
Linear Primer Extension Reactions	37	(2)
High-Density Labeling	39	(2)
Exonuclease Degradation	41	(2)
Restriction Enzyme Cutting and DNA Sizing	43	(3)
Polymerase Chain Reactions	46	(13)
FCS Autocorrelation Analysis: New Detection Methods	47	(6)
FCS Cross-Correlation Analysis: A New Concept for PCR	53	(6)
Summary and Conclusions	59	(6)
References	60	(5)
Strain-Dependent Fluorescence Correlation Spectroscopy: Proposing a New Measurement for Conformational Fluctuations of Biological Macromolecules	65	(19)
Hong Qian
Elliot L. Elson
Introduction	65	(2)
Theory	67	(3)
A Simple Example	70	(6)
Discussion	76	(6)
SD-FCS	76	(4)
Comparison of SD-FCS with Conventional FCS	80	(1)
Applications and Feasibility	81	(1)
Summary	82	(2)
References	82	(2)
Applications of FCS to Protein-Ligand Interactions: Comparison with Fluorescence Polarization	84	(17)
Edmund Matayoshi
Kerry Swift
Fluorescence Polarization versus FCS	84	(3)
Experimental Methods	87	(1)
HIV Protease	88	(2)
Death Domain Interactions	90	(3)
Antibody-Small Ligand Interactions	93	(2)
Antibody-Large Ligand Interactions	95	(1)
Conclusions	96	(5)
References	97	(4)
Part II FCS at the Cellular Level
FCS-Analysis of Ligand-Receptor Interactions in Living Cells	101	(31)
Aladdin Pramanik
Rudolf Rigler
Introduction	101	(1)
Materials and Methods	101	(7)
Chemicals	101	(1)
Cell Culture	102	(1)
Fluorescence Correlation Spectroscopy (FCS)	102	(6)
Results	108	(14)
Background Signal	108	(1)
Binding of Rh-Ligands to the Cell Membranes	108	(5)
Presentation of Ligand-Receptor Complexes with Distribution of Diffusion Times	113	(2)
Saturation of Binding	115	(2)
Specificity and Kinetics of Binding	117	(3)
Measurement of the Association Rate Constant	120	(1)
Effect of Pertussis Toxin on the Ligand Binding	121	(1)
Measurement of IC50	121	(1)
Discussion	122	(6)
Demonstration of Specific Binding	122	(1)
Nature of Ligand-Receptor Interaction	123	(1)
Binding Kinetics	124	(1)
Different Ligand-Receptor Complexes and Binding Sites/Receptor Subtypes	124	(1)
Allosteric Nature of Signal Transduction and Receptor Aggregation	125	(2)
Problems, Limitations, and Precautions	127	(1)
Future Perspectives and Cross-Correlation	128	(4)
References	129	(3)
Fluorescence Correlation Microscopy (FCM): Fluorescence Correlation Spectroscopy (FCS) in Cell Biology	132	(30)
Roland Brock
Thomas M. Jovin
Introduction	132	(1)
Theory of Cellular FCS	133	(5)
FCS in Multi-component Systems	133	(1)
Detection of Molecular Association Without Explicit Analysis of the Diffusion Constant D	134	(1)
Determination of N for Distributions of Molecules Carrying Different Numbers of Fluorophores per Molecule	135	(1)
Intracellular FCS - Approximation of Local Equilibria	136	(1)
FCS in Small Volumes-The Problem of Fluorophore Depletion	137	(1)
FCS-Derived Parameters in Cell Biology	137	(1)
Instrumental Requirements for Intracellular FCS	138	(4)
Design of the Fluorescence Correlation Microscope	138	(4)
Applications of Intracellular FCM	142	(11)
FCM in the Analysis of Receptor Diffusion-Measurement Protocols for Intracellular FCM	142	(6)
FCM in the Analysis of Metabolic Conversions	148	(3)
Comparison of Cytoplasmic and Nuclear GFP	151	(1)
FCM in Cellular High Throughput Screening	152	(1)
Limitations and Perspectives of Cellular FCM	153	(9)
FCM-Specific Problems in Intracellular Research	153	(2)
Perspectives in Cellular FCM	155	(2)
Comparison of FCM with Other Techniques	157	(2)
References	159	(3)
FCS and Spatial Correlations on Biological Surfaces	162	(25)
Nils O. Petersen
The Problem	162	(1)
The Solution	163	(2)
The Experiment	165	(5)
Generating Images Using a Confocal Microscope	165	(1)
Correlation Calculations	165	(1)
Correlation Function Analysis	166	(1)
Extracting the Amplitude Information	167	(2)
Technical Issues	169	(1)
Interpretation of Correlation Function Amplitudes	170	(4)
Cluster Densities	170	(1)
Degree of Aggregation	171	(1)
Multiple Populations	172	(1)
Dynamics of Aggregation	173	(1)
Intermolecular Interactions and Colocalization	173	(1)
Applications to Cell Surfaces	174	(7)
Receptor Distributions	175	(3)
Interactions in Coated Pits	178	(2)
Virus Assembly and Fusion	180	(1)
Other Applications and Future Prospects	181	(1)
Conclusions	181	(6)
References	183	(4)
Part III Applications in Biotechnology, Drug Screening, and Diagnostics
Dual-Color Confocal Fluorescence Spectroscopy and its Application in Biotechnology	187	(17)
Andre Koltermann
Ulrich Kettling
Jens Stephan
Thorsten Winkler
Manfred Eigen
Introduction	187	(2)
Real-Time Monitoring of Enzymatic Activity by Dual-Color FCS	189	(4)
RAPID FCS and CFCA for Screening Applications	193	(6)
Applications in Evolutionary Biotechnology	199	(2)
Outlook	201	(3)
References	202	(2)
Nanoparticle Immunoassays: A new Method for Use in Molecular Diagnostics and High Throughput Pharmaceutical Screening based on Fluorescence Correlation Spectroscopy	204	(21)
F.J. Meyer-Almes
Introduction	204	(2)
Theory	206	(4)
Competitive NPIA	206	(1)
Sandwich NPIA	207	(2)
Autocorrelation Amplitudes	209	(1)
Material and Methods	210	(2)
Substances	210	(1)
Equipment	211	(1)
Reactions	211	(1)
Simulations and Data Fitting	212	(1)
Results	212	(9)
Simulations	212	(1)
Experiments	213	(8)
Discussion	221	(4)
References	223	(2)
Protein Aggregation Associated with Alzheimer and Prion Diseases	225	(26)
Detlev Riesner
Introduction	225	(1)
Prion-Protein Multimerization	226	(11)
Conformation and State of Aggregation	226	(3)
Analysis of Multimerization by FCS	229	(2)
Influence of Fluorescence Labeling on the Multimerization Reaction	231	(2)
Kinetics of Spontaneous Multimerization	233	(1)
Seeded Multimerization of PrP	233	(3)
Summary of PrP Conformational Transitions	236	(1)
Amyloid β-Peptide Multimerization	237	(7)
Spontaneous Multimerization	237	(3)
Seeded Aggregation as a Diagnostic Tool	240	(4)
Synopsis	244	(7)
References	245	(6)
Part IV Environmental Analysis and Monitoring
Application of FCS to the Study of Environmental Systems	251	(25)
Konstantin Starchev
Kevin J. Wilkinson
Jacques Buffle
Introduction	251	(1)
Nature and Characteristics of Aquatic and Terrestrial Colloids and Biopolymers	252	(8)
Nature of the Major Aquatic and Terrestrial Colloids	252	(5)
Aggregation Processes and Aggregate Structure	257	(2)
Potential Advantages and Limitations of FCS for Environmental Applications	259	(1)
Development of FCS for its Application to the Study of Environmental Systems	260	(6)
Colloids With Sizes Comparable to the Beam Width	260	(2)
Polydisperse Systems	262	(4)
Example: Determination of the Diffusion Coefficients of Humic Substances as a Function of Solution Conditions	266	(7)
Factors Distinguishing Humic Substances From Model Compounds	268	(3)
The Role of Solution Conditions (pH, Ionic Strength, Concentration) on the Diffusion Coefficients of Humic Substances	271	(2)
Conclusions and Future Perspectives	273	(3)
References	274	(2)
Photophysical Aspects of FCS Measurements	276	(29)
Jerker Widengren
Introduction	276	(2)
Photophysics in the Fast Time Range	278	(16)
Triplet State Formation	281	(6)
Charge Transfer Reactions	287	(3)
Photo-Induced Isomerization	290	(3)
Effects of Non-Uniform Excitation	293	(1)
Photophysics in the Slow Time Range-Photodegradation	294	(2)
Strategies to Improve Photophysical Conditions	296	(3)
Concluding Remarks	299	(6)
References	300	(5)
Part V New Developments and Trends
Fluorescence Correlation Spectroscopy: Genesis, Evolution, Maturation and Prognosis	305	(26)
Watt W. Webb
Introduction	305	(1)
Genesis	305	(5)
Evolution	310	(4)
Maturation of FCS at Cornell	314	(12)
Green Fluorescent Proteins in FCS	315	(4)
Molecular Diffusion in Lipid Membranes of Giant Unilamellar Vesicles	319	(2)
Two-Photon Molecular Excitation (2PE) for FCS	321	(2)
FCS in Cells and Tissues	323	(3)
Prognosis for FCS	326	(5)
References	328	(3)
ConfoCor 2 The Second Generation of Fluorescence Correlation Microscopes	331	(15)
Tilo Jankowski
Reinhard Janka
Introduction	331	(1)
Instrumental Setup	331	(8)
Laser Module	332	(1)
Detection Unit	333	(2)
Detection Efficiency Profile	335	(2)
Detection of Fuorescence Correlation Signals	337	(1)
FCS Data Analysis	338	(1)
Autocorrelation Measurements	339	(3)
Cross Correlation Measurements	342	(3)
Summary	345	(1)
References	345	(1)
Antibunching and Rotational Diffusion in FCS	346	(14)
Ulo Mets
Introduction	346	(1)
Antibunching	346	(8)
Rotational Diffusion	354	(4)
Discussion	358	(2)
References	359	(1)
Cross-correlation analysis in FCS	360	(19)
Petra Schwille
Introduction	360	(3)
Theory	363	(8)
Fluctuation Correlations	363	(1)
The Effective Measurement Volume in FCS	364	(2)
Autocorrelation and Cross-correlation Functions for Pure Diffusion	366	(2)
Detector Cross-Talk	368	(1)
Not Completely Overlapping Detection Volumes	369	(1)
Cross-correlation of Internal Fluctuations	370	(1)
Experimental Realization	371	(2)
Applications	373	(6)
Slow Association Reactions: Comparison Between Autocorrelation and Cross-correlation	373	(2)
Cross-correlation Applications in Various Biochemical Systems	375	(2)
Outlook	377	(1)
References	377	(2)
Cross-correlated Flow Analysis in Microstructures	379	(17)
Michael Brinkmeier
Introduction	379	(1)
The Experimental Setup	380	(2)
Theory	382	(5)
Pseudo-Autocorrelation	386	(1)
Experimental Procedures	387	(3)
Optimizing the Setup	387	(1)
Flow Measurements	387	(3)
Applications	390	(4)
Continuous Flow Kinetics	391	(2)
Rapid DNA Sequencing	393	(1)
Conclusion	394	(2)
References	395	(1)
Introduction to the Theory of Fluorescence Intensity Distribution Analysis	396	(14)
Peet Kask
Kaupo Palo
Introduction	396	(2)
Photon Count Number Distribution Corresponding to a Rectangular Sample Profile	398	(1)
Photon Count Number Distribution Corresponding to an Arbitrary Sample Profile: The Convolution Technique	398	(1)
Photon Count Number Distribution Corresponding to an Arbitrary Sample Profile: The Technique of the Generating Function	399	(1)
Sample Profile Models	400	(1)
Distribution of the Specific Brightness Within a Species	401	(1)
Weighting in FIDA	402	(1)
Data Simulation Algorithms	403	(1)
Statistical Errors of Estimated Parameters	404	(6)
References	409	(1)
Photon Counting Histogram Statistics	410	(28)
Joachim D. Muller
Yan Chen
Enrico Gratton
Introduction	410	(1)
Theory	411	(1)
PCH and the Theory of Photon Detection	411	(10)
PCH of a Single Particle	414	(1)
PCH of Multiple Particles	414	(1)
PCH of Particles with Number Fluctuations	415	(1)
PCH of Multiple Species	416	(1)
PCH for Different PSFs	416	(2)
Describing PCH with the Moment Generating Function	418	(2)
Two-fold PCH Statistics	420	(1)
Data Analysis	421	(1)
Single Species PCH	421	(7)
Influence of the Particle Concentration	421	(2)
Influence of Molecular Brightness	423	(2)
Sensitivity of PCH Algorithm	425	(3)
PCH for Multiple Species	428	(5)
Resolvability of Two Species	428	(2)
Experimental Results	430	(3)
Conclusions	433	(5)
References	435	(3)
High Order Autocorrelation in Fluorescence Correlation Spectroscopy	438	(21)
Nancy L. Thompson
Jennifer L. Mitchell
Introduction	438	(1)
Temporal High Order FCS	439	(12)
Definitions	439	(1)
First Order Fluorescence Fluctuation Autocorrelation	440	(2)
High Order Fluorescence Fluctuation Autocorrelation	442	(2)
Multicomponent Analysis	444	(3)
Experimental Considerations	447	(2)
Experimental Applications	449	(2)
Spatial High Order FCS	451	(4)
Overview	451	(1)
Spatial Fluorescence Fluctuation Autocorrelation Functions	452	(1)
Autocorrelation Function Magnitudes and Decay Shapes	452	(1)
Experimental Considerations	453	(1)
Experimental Application	454	(1)
Discussion	455	(4)
References	456	(3)
FCS in Single Molecule Analysis	459	(18)
R. Rigler
S. Wennmalm
L. Edman
Introduction	459	(1)
Single Molecule Detection in Solution and Correlation Functions	459	(3)
Confocal Single Molecule Imaging	462	(1)
Conformatial Transitions in Single DNA Molecules	463	(1)
Single Molecule Traces	464	(1)
Homogeneous and Heterogeneous Behavior	465	(1)
Time Resolution of Single Molecule Behaviour	465	(2)
Kinetic Analysis, Death Numbers, and Survival Times	467	(1)
The Fluctuating Enzyme	468	(1)
Evidence for Multiple Conformational Transition and Catalysis	468	(6)
Higher Order Correlations and Non-Markovian Behavior	474	(1)
Conclusions	474	(3)
References	475	(2)
Subject Index	477