Single Molecule Tools, Part B: Super-Resolution, Particle Tracking, Multiparameter, and Force Based Methods

1. Auflage 2010
ISBN: 978-0-12-381483-8
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, Band 475, 720 Seiten

Reihe: Methods in Enzymology

Single molecule tools have begun to revolutionize the molecular sciences, from biophysics to chemistry to cell biology. They hold the promise to be able to directly observe previously unseen molecular heterogeneities, quantitatively dissect complex reaction kinetics, ultimately miniaturize enzyme assays, image components of spatially distributed samples, probe the mechanical properties of single molecules in their native environment, and 'just look at the thing' as anticipated by the visionary Richard Feynman already half a century ago. This volume captures a snapshot of this vibrant, rapidly expanding field, presenting articles from pioneers in the field intended to guide both the newcomer and the expert through the intricacies of getting single molecule tools.
* Includes time-tested core methods and new innovations applicable to any researcher employing single molecule tools * Methods included are useful to both established researchers and newcomers to the field * Relevant background and reference information given for procedures can be used as a guide to developing protocols in a number of disciplines

Autoren/Hrsg.

Inhaltsverzeichnis

1;Front Cover;1
2;Methods in Enzymology: Single Molecule Tools,Part B: Super-Resolution,Particle Tracking,Multiparameter, andForce Based Methods;4
3;Copyright Page;5
4;Contents;6
5;Contributors;14
6;Preface;22
7;Volume in Series;24
8;Chapter 1: Super-Accuracy and Super-Resolution: GettingAround the Diffraction Limit;52
8.1;1. Overview: Accuracy and Resolution;53
8.2;2. Getting Super-Accuracy;55
8.3;3. Calculating Super-Accuracy;58
8.4;4. Reaching Super-Resolution;64
8.5;5. Future Directions;73
8.6;References;73
9;Chapter 2: Molecules and Methods for Super-Resolution Imaging;78
9.1;1. Introduction;79
9.2;2. Molecules for Super-Resolution Imaging;81
9.3;3. Selected Methods for Super-Resolution Imaging;97
9.4;Acknowledgments;106
9.5;References;106
10;Chapter 3: Tracking Single Proteins in Live Cells Using Single-Chain Antibody Fragment-Fluorescent Quantum Dot Affinity Pair;112
10.1;1. Introduction;113
10.2;2. The Method: Targeting QDs via a Single-Chain Variable Fragment-Hapten Pair;115
10.3;3. Functionalization of QDs;117
10.4;4. Quantification of the Number of FL Molecules per FL-pc-QD;120
10.5;5. Binding of FL-QDs to Anti-scFv Fusion Constructs;122
10.6;6. DNA Constructs for Single FL-QD Imaging in Live Cells ;124
10.7;7. Single-Molecule Imaging of Live Mammalian Cells;125
10.8;Acknowledgments;126
10.9;References;128
11;Chapter 4: Recording Single Motor Proteins in the Cytoplasm of Mammalian Cells;132
11.1;1. Introduction;133
11.2;2. Basic Principles;135
11.3;3. Labeling Molecular Motors for In Vivo Observations;136
11.4;4. Instrumentation for Tracking Single Motors In Vivo;140
11.5;5. Detailed Experimental Procedures;146
11.6;6. Summary and Conclusions;154
11.7;Acknowledgments;154
11.8;References;155
12;Chapter 5: Single-Particle Tracking Photoactivated Localization Microscopy for Mapping Single-Molecule Dynamics;160
12.1;1. Introduction;161
12.2;2. Description of the sptPALM Method;162
12.3;3. Labeling with Photoactivatable Fluorescent Probes;164
12.4;4. Tracking Single Molecules;165
12.5;5. Experimental Example: sptPALM on a Membrane Protein;167
12.6;6. Conclusions;169
12.7;References;169
13;Chapter 6: A Bird's Eye View: Tracking SlowNanometer-Scale Movements ofSingle Molecular Nano-assemblies;172
13.1;1. Introduction;173
13.2;2. DNA-Based Nanowalkers;176
13.3;3. Considerations for Fluorescence Imaging of Slowly Moving Particles;180
13.4;4. Single-Molecule Fluorescence Tracking of Nanowalkers;182
13.5;5. Extracting Super-Resolution Position Information;187
13.6;6. Concluding Remarks;194
13.7;Acknowledgments;195
13.8;References;195
14;Chapter 7: Anti-Brownian Traps for Studies on Single Molecules;200
14.1;1. Theoretical Overview;201
14.2;2. Anti-Brownian Trapping Systems;206
14.3;3. The ABEL Trap;212
14.4;4. Applications;220
14.5;5. Future Work: En Route to Single Fluorophores;221
14.6;Acknowledgments;222
14.7;References;222
15;Chapter 8: Plasmon Rulers as Dynamic Molecular Rulers in Enzymology;226
15.1;1. Introduction;227
15.2;2. The Basic Idea: Distance Dependence of Plasmon Coupling;228
15.3;3. Hardware Needed for Single Particle Rayleigh Scattering Spectroscopy;230
15.4;4. Which Readout-Intensity, Polarization, or Color?;232
15.5;5. Ruler Calibration?;233
15.6;6. Plasmon Ruler Assembly and Purification;235
15.7;7. Example 1: Dynamics of DNA Bending and Cleavage by Single EcoRV Restriction Enzymes;237
15.8;8. Example 2: Spermidine Modulated Ribonuclease Activity Probed by RNA Plasmon Rulers;243
15.9;9. Outlook;245
15.10;References;247
16;Chapter 9: Quantitative Analysis of DNA-Looping Kinetics from Tethered Particle Motion Experiments;250
16.1;1. Introduction;251
16.2;2. Change-Point Algorithm;252
16.3;3. Data Clustering and Expectation-Maximization Algorithm;254
16.4;4. Adaptation of the Method to the Case of TPM Data Analysis;255
16.5;5. Performance of the Method;260
16.6;6. Comparison with the Threshold Method;263
16.7;7. Application to TPM Experiments: CI-Induced Looping in lambda-DNA ;266
16.8;8. Conclusions;268
16.9;Acknowledgments;269
16.10;References;269
17;Chapter 10: Methods in Statistical Kinetics;272
17.1;1. Introduction;273
17.2;2. The Formalism of Statistical Kinetics;274
17.3;3. Characterizing Fluctuations;283
17.4;4. Extracting Mechanistic Constraints from Moments;291
17.5;5. Conclusions and Future Outlook;299
17.6;Appendix;300
17.7;References;306
18;Chapter 11: Visualizing DNA Replication at the Single-Molecule Level;310
18.1;1. Introduction;311
18.2;2. Observing Replication Loops with Tethered Bead Motion;312
18.3;3. Fluorescence Visualization of DNA Replication;322
18.4;Acknowledgments;328
18.5;References;328
19;Chapter 12: Measurement of the Conformational State of F1-ATPase by Single-Molecule Rotation;330
19.1;1. Introduction;331
19.2;2. Sample Preparation;333
19.3;3. Single-Molecule Cross-Link Experiment;338
19.4;4. Pausing with AMP-PNP or/and N3-;344
19.5;Acknowledgments;346
19.6;References;346
20;Chapter 13: Magnetic Tweezers for the Study of DNA Tracking Motors;348
20.1;1. Introduction;349
20.2;2. Experimental Setup;350
20.3;3. Methods and Protocols;351
20.4;4. Application to the Study of FtsK;359
20.5;5. Application to the Study of the GP41 Helicase;363
20.6;6. Conclusions;369
20.7;Acknowledgments;370
20.8;References;370
21;Chapter 14: Single-Molecule Dual-Beam Optical Trap Analysis of Protein Structure and Function;372
21.1;1. Introduction;373
21.2;2. Insights into Myosin Function Using a Dual-Beam Optical Trap;373
21.3;3. Optical Trap Instrumentation;375
21.4;4. Optical Trapping Experiment;409
21.5;5. Data Analysis;412
21.6;6. Conclusion;420
21.7;Appendix;420
21.8;Acknowledgments;423
21.9;References;423
22;Chapter 15: An Optical Apparatus for Rotation and Trapping;428
22.1;1. Introduction;429
22.2;2. Optical Trapping and Rotation of Microparticles;430
22.3;3. The Instrument;436
22.4;4. Fabrication of Anisotropic Particles;441
22.5;5. Instrument Calibration;446
22.6;6. Simultaneous Application of Force and Torque Using Optical Tweezers;451
22.7;7. Conclusions;453
22.8;Acknowledgments;453
22.9;References;454
23;Chapter 16: Force-Fluorescence Spectroscopy at the Single-Molecule Level;456
23.1;1. Introduction;457
23.2;2. Setup;458
23.3;3. Optical Trapping;461
23.4;4. Fluorescence Detection;464
23.5;5. Coalignment of Confocal and Optical Trapping;467
23.6;6. Sample Preparation Protocols;469
23.7;7. Applications to Biological Systems;474
23.8;8. Outlook;474
23.9;Acknowledgments;475
23.10;References;475
24;Chapter 17: Combining Optical Tweezers, Single-Molecule Fluorescence Microscopy, and Microfluidics for Studies of DNA-Protein Interactions;478
24.1;1. Introduction;479
24.2;2. Instrumentation;481
24.3;3. Preparation of Reagents;494
24.4;4. Combining Optical Trapping, Fluorescence Microscopy, and Microfluidics: Example Protocols;496
24.5;5. Conclusions;501
24.6;Acknowledgments;502
24.7;References;502
25;Chapter 18: Accurate Single-Molecule FRET Studies Using Multiparameter Fluorescence Detection;506
25.1;1. Introduction;507
25.2;2. FRET Theory;512
25.3;3. Fluorescence Properties and Measurement Techniques;517
25.4;4. Qualitative Description of smFRET;525
25.5;5. Quantitative Description of smFRET;540
25.6;6. Discussion;554
25.7;Acknowledgments;556
25.8;References;557
26;Chapter 19: Atomic Force Microscopy Studies of Human Rhinovirus: Topologyand Molecular Forces;566
26.1;1. Introduction;567
26.2;2. Results and Discussion;568
26.3;References;587
27;Chapter 20: High-Speed Atomic Force Microscopy Techniques for Observing Dynamic Biomolecular Processes;592
27.1;1. Introduction;593
27.2;2. Survey of Requirements for High-Speed Bio-AFM Imaging;594
27.3;3. Substrate Surfaces;595
27.4;4. Control of Diffusional Mobility;605
27.5;5. Protein 2D Crystals as Targets to Study;606
27.6;6. Low-Invasive Imaging;609
27.7;7. UV Flash-Photolysis of Caged Compounds;610
27.8;8. Cantilever Tip;612
27.9;References;613
28;Chapter 21: Nanopore Force Spectroscopy Tools for Analyzing Single Biomolecular Complexes;616
28.1;1. Introduction;617
28.2;2. The Nanopore Method;618
28.3;3. DNA Unzipping Kinetics Studied Using Nanopore Force Spectroscopy;628
28.4;4. Conclusions and Summary;636
28.5;Acknowledgments;638
28.6;References;638
29;Chapter 22: Analysis of Single Nucleic Acid Molecules with Protein Nanopores;642
29.1;1. Background: Analysis of Nucleic Acids with Nanopores;644
29.2;2. Electrical Recording with Planar Lipid Bilayers;652
29.3;3. Nanopores;661
29.4;4. Materials;664
29.5;5. Data Acquisition and Analysis;667
29.6;References;670
30;Author Index;676
31;Subject Index;700
32;Color Plate;716