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

Super-Accuracy and Super-Resolution: Getting Around the Diffraction Limit
......Page 41
Overview: Accuracy and Resolution
......Page 42
Getting Super-Accuracy
......Page 44
Calculating Super-Accuracy
......Page 47
Kinesin walks hand-over-hand
......Page 48
FIONA without fluorescence
......Page 50
FIONA in a live organism
......Page 52
Reaching Super-Resolution
......Page 53
Single-molecule high-resolution imaging with photobleaching
......Page 54
Single-molecule high-resolution colocalization
......Page 55
PALM and STORM
......Page 57
Stimulated emission depletion microscopy (STED)
......Page 59
References......Page 62
Molecules and Methods for Super-Resolution Imaging
......Page 67
Introduction
......Page 68
Strategies for designing and characterizing photoactivatable fluorophores
......Page 70
Photobleaching and photoconversion quantum yields
......Page 71
Effective turn-on ratio
......Page 72
The azido-DCDHF class of photoswitchable fluorophores
......Page 74
Design
......Page 78
Photoreaction characterization
......Page 79
Synthesis and characterization of covalent Cy3-Cy5 heterodimers
......Page 80
Synthesis
......Page 81
Bulk photophysical properties
......Page 82
EYFP as a photoswitchable emitter
......Page 83
Sample preparation
......Page 86
Image reconstruction
......Page 87
Time-lapse imaging
......Page 89
Three-dimensional single-molecule imaging using double-helix photoactivated localization microscopy
......Page 90
References......Page 95
Tracking Single Proteins in Live Cells Using Single-Chain Antibody Fragment-Fluorescent Quantum Dot Affinity Pair
......Page 100
Introduction
......Page 101
The Method: Targeting QDs via a Single-Chain Variable Fragment-Hapten Pair
......Page 103
Peptide coating of QDs
......Page 105
Varying the stoichiometry of FL-pc-QDs
......Page 107
Quantification of the Number of FL Molecules per FL-pc-QD
......Page 108
Quantification of the binding affinity of FL-QD to α-FL scFv
......Page 110
DNA Constructs for Single FL-QD Imaging in Live Cells
......Page 112
Single-Molecule Imaging of Live Mammalian Cells
......Page 113
Acknowledgments......Page 114
References......Page 116
Recording Single Motor Proteins in the Cytoplasm of Mammalian Cells
......Page 119
Introduction
......Page 120
Basic Principles
......Page 122
Organelles as labels for cytoplasmic motors
......Page 123
Organic fluorophores and genetic tags
......Page 125
Instrumentation for Tracking Single Motors In Vivo
......Page 127
Multicolor TIRF illumination
......Page 128
Dark-field excitation to track nanoparticles
......Page 130
Recording single-molecule events
......Page 131
Postimage processing
......Page 132
Detailed Experimental Procedures
......Page 133
Fluorescent protein fusion plasmids
......Page 134
Cell culture
......Page 135
Recording and analyzing single-molecule in vivo events
......Page 137
Identifying individual single motor events in live cells
......Page 138
SD maps
......Page 139
High-resolution tracking
......Page 140
Acknowledgments......Page 141
References......Page 142
Single-Particle Tracking Photoactivated Localization Microscopy for Mapping Single-Molecule Dynamics......Page 146
Introduction......Page 147
Description of the sptPALM Method......Page 148
Photoactivatable fluorescent proteins......Page 150
Molecule identification......Page 151
Tracking algorithms......Page 152
Experimental Example: sptPALM on a Membrane Protein......Page 153
References......Page 155
A Bird's Eye View: Tracking Slow Nanometer-Scale Movements of Single Molecular Nano-assemblies
......Page 158
Introduction......Page 159
History of fluorescent single-particle tracking......Page 160
The molecular ``spider´´......Page 162
Behavior-based molecular robots: Programming tasks into the walker's environment......Page 164
Prolonging fluorophore lifetime......Page 166
Control for stage and focal drift......Page 167
Slide preparation......Page 168
Experimental procedure......Page 170
Instrumentation for imaging......Page 171
Mapping the two emission channels onto one another......Page 173
Quality control......Page 174
Displacement plots......Page 176
Ensemble-averaged mean-square displacement plots......Page 178
Concluding Remarks......Page 180
References......Page 181
Anti-Brownian Traps for Studies on Single Molecules
......Page 186
Single-molecule spectroscopy yields important dynamical information but is hampered by Brownian motion
......Page 187
Tracking and feedback
......Page 189
Anti-Brownian Trapping Systems
......Page 192
Camera
......Page 193
Multiphotodiode
......Page 194
Scanning
......Page 195
Feedback systems
......Page 196
Electrokinetic feedback
......Page 197
The ABEL Trap
......Page 198
Photon-by-photon feedback
......Page 200
Illumination system
......Page 201
Tracking and feedback system
......Page 202
Kalman filter signal-processing
......Page 203
Microfluidics
......Page 205
Applications
......Page 206
Future Work: En Route to Single Fluorophores
......Page 207
References......Page 208
Plasmon Rulers as Dynamic Molecular Rulers in Enzymology......Page 212
Introduction......Page 213
The Basic Idea: Distance Dependence of Plasmon Coupling......Page 214
Hardware Needed for Single Particle Rayleigh Scattering Spectroscopy......Page 216
Intensity......Page 218
Ruler Calibration?......Page 219
Plasmon Ruler Assembly and Purification......Page 221
Example 1: Dynamics of DNA Bending and Cleavage by Single EcoRV Restriction Enzymes......Page 223
Example 2: Spermidine Modulated Ribonuclease Activity Probed by RNA Plasmon Rulers......Page 229
Outlook......Page 231
References......Page 233
Quantitative Analysis of DNA-Looping Kinetics from Tethered Particle Motion Experiments......Page 236
Introduction......Page 237
Change-Point Algorithm......Page 238
Data Clustering and Expectation-Maximization Algorithm......Page 240
Adaptation of the Method to the Case of TPM Data Analysis......Page 241
Performance of the Method......Page 246
Comparison with the Threshold Method......Page 249
Application to TPM Experiments: CI-Induced Looping in λ-DNA
......Page 252
Conclusions......Page 254
References......Page 255
Methods in Statistical Kinetics
......Page 258
Introduction
......Page 259
From steady-state kinetics to statistical kinetics
......Page 260
Basic statistics of the cycle completion time
......Page 263
The ``memory-less´´ enzyme
......Page 264
Lifetime statistics
......Page 266
State visitation statistics
......Page 267
Fitting distributions
......Page 269
Calculating moments
......Page 272
Multiple pathways and multiple steps
......Page 273
Extracting Mechanistic Constraints from Moments
......Page 277
The randomness parameter and nmin
......Page 278
Classifying fluctuations
......Page 279
Mechanistic constraints
......Page 281
Conclusions and Future Outlook
......Page 285
Calculating the moments of the cycle completion time
......Page 286
Estimation of systematic errors in statistical moments
......Page 288
References......Page 292
Visualizing DNA Replication at the Single-Molecule Level
......Page 295
Introduction
......Page 296
Functionalizing glass coverslips
......Page 297
Functionalizing polystyrene beads
......Page 298
Preparation of forked λ-DNA substrate 
......Page 299
Construction of flow chamber
......Page 301
Replication reaction and imaging
......Page 304
Data analysis
......Page 305
Preparation of rolling-circle template
......Page 307
Construction of flow chamber
......Page 308
Replication reaction and imaging
......Page 309
Data analysis
......Page 311
References......Page 313
Measurement of the Conformational State of F1-ATPase by Single-Molecule Rotation......Page 315
F1-ATPase......Page 316
Conformations of F1 found in crystal structures and single-molecule studies......Page 317
Design of a cross-link mutant......Page 318
Biochemical assay for the β-γ cross-link
......Page 320
Preparation of a hybrid F1......Page 321
Rotation assay......Page 323
Identification in the rotation assay of the hybrid F1 with one β(E190D/E391C)
......Page 324
Cross-link formation in the rotation assay......Page 325
Analysis of pause positions of the cross-linked α3β3γ subcomplex......Page 327
Pausing with AMP-PNP or/and N3-......Page 329
References......Page 331
Magnetic Tweezers for the Study of DNA Tracking Motors
......Page 333
Introduction
......Page 334
Experimental Setup
......Page 335
Surface preparation
......Page 336
DNA hairpin
......Page 337
dsDNA construct
......Page 340
Selection of beads tethered by a single DNA hairpin
......Page 342
Selection of beads tethered by a coilable DNA molecule
......Page 343
Application to the Study of FtsK
......Page 344
FtsK activity
......Page 345
Force-extension curve
......Page 348
Detecting helicase activity
......Page 349
Optimizing helicase loading
......Page 350
Measuring unwinding and ssDNA translocation activities
......Page 352
Conclusions
......Page 354
References......Page 355
Single-Molecule Dual-Beam Optical Trap Analysis of Protein Structure and Function
......Page 357
Insights into Myosin Function Using a Dual-Beam Optical Trap
......Page 358
Basic concept of an optical trap
......Page 360
Design and components
......Page 361
Objective (OBJ)
......Page 368
Beam steering for slow and coarse positioning
......Page 374
Detection of bead position
......Page 375
Noise and stability considerations
......Page 377
Air fluctuation
......Page 378
Electrical noise of data acquisition
......Page 379
Alignment protocol
......Page 380
Conversion of changes in the voltage input to the AODs to bead displacement
......Page 392
Measuring trap stiffness
......Page 393
Forming an actin dumbbell
......Page 394
Testing platforms
......Page 395
Measurement of dwell time
......Page 397
Graphing the data
......Page 398
Maximum likelihood estimation
......Page 399
The bootstrap method
......Page 400
Measurement of stroke size: Compliance correction for nonprocessive motors
......Page 401
Strategy for compliance correction in a dual-beam optical trap
......Page 403
Alignment for the feedback control of a single beam by the AOD
......Page 405
Correlation coefficient between two trapped beads (CorrCoef)
......Page 406
References......Page 408
An Optical Apparatus for Rotation and Trapping......Page 412
Introduction......Page 413
The principles of optical manipulation......Page 414

Sources of particle anisotropy......Page 417

Overview......Page 420
The microscope......Page 422
Signal detection and processing......Page 424
Particles with shape asymmetry......Page 425
Particles with optical asymmetry......Page 426
Protocol......Page 427
Standard optical tweezers calibration methods......Page 430
Force calibration......Page 431

Implementation of an optical torque clamp......Page 433
Twisting single DNA molecules under tension......Page 435
Acknowledgments......Page 437
References......Page 438
Force-Fluorescence Spectroscopy at the Single-Molecule Level......Page 440
Introduction......Page 441
Optical scheme......Page 442
Surface-tethered assays......Page 444
Determination of the trapping height......Page 445
Position detector calibration......Page 446
Determination of the trap stiffness......Page 447
Single-molecule confocal microscopy......Page 448
General methods for data analysis......Page 449
Calibration of piezo-controlled mirror......Page 451
Nucleic acid and protein labeling......Page 453
Polymer-passivated surface preparation......Page 454
DNA/RNA sample preparation......Page 455
Preparation of antidigoxigenin-coated microspheres......Page 456
Sample assembly......Page 457
Outlook......Page 458
References......Page 459
Combining Optical Tweezers, Single-Molecule Fluorescence Microscopy, and Microfluidics for Studies of DNA-Protein Interactions
......Page 462
Introduction
......Page 463
Principles of optical trapping
......Page 465
Light sources for optical tweezers
......Page 466
Generation and steering of a dual trap
......Page 468
Laser detection and optical trap calibration
......Page 470
Single-molecule fluorescence microscopy and background reduction
......Page 472
Photobleaching
......Page 474
Microfluidics
......Page 475
Example protocol I: Biotinylation of lambda DNA
......Page 478
Fluorescent labeling of proteins
......Page 479
Combining Optical Trapping, Fluorescence Microscopy, and Microfluidics: Example Protocols
......Page 480
Example protocol II: Sequential isolation and visualization of a single DNA molecule using YOYO-1
......Page 481
Example protocol IV: Binding of Rad51 to dsDNA
......Page 483
Conclusions
......Page 485
References......Page 486
Accurate Single-Molecule FRET Studies Using Multiparameter Fluorescence Detection
......Page 489
Introduction......Page 490
Basic FRET theory in a nutshell......Page 495
The orientation factor kappa2......Page 499
Timescales of biomolecular processes and fluorescence techniques......Page 500
Dimensions of fluorescence......Page 502
Experimental setup and data registration......Page 504
Improving accuracy of smFRET measurements......Page 507
Intensity measurements......Page 508
Fluorescence lifetime measurements......Page 510
Two-dimensional FRET analysis......Page 511
Anisotropy versus donor lifetime......Page 514
Example: HIV-1 reverse transcriptase (RT)......Page 515
Donor quenching......Page 516
Dynamic behavior......Page 519
Example: Conformational dynamics of syntaxin-1 (Sx)......Page 521
Possible artifacts......Page 522
Basic theory of photon distribution analysis (PDA)......Page 523
FRET analysis for acceptors with additional direct excitation......Page 525
Shot-noise limited FRET signal distributions and additional broadening......Page 526
PDA of dynamic systems......Page 528
gG/gR ratio for spectral sensitivity......Page 530
Display of FRET indicators......Page 531
High-resolution FRET structures......Page 532
Improving accuracy of FRET-based structures......Page 533
Example: DNA structure determination......Page 534
MFD 2D analysis......Page 537
PDA......Page 538
Acknowledgments......Page 539
References......Page 540
Atomic Force Microscopy Studies of Human Rhinovirus: Topology and Molecular Forces
......Page 549
Introduction
......Page 550
Protocol: Human rhinovirus production and isolation
......Page 551
Preparation of human His6-tagged MBP-very-low-density lipoprotein receptors
......Page 552
AFM imaging setting
......Page 553
Protocol: MACmode AFM imaging
......Page 554
Protocol: HRV2 immobilization onto mica
......Page 555
Crystalline arrangement of HRV2 on model cell membranes
......Page 557
Imaging of receptor binding to the virus
......Page 560
Protocol: Binding of receptors to HRV2 immobilized on mica
......Page 561
Force spectroscopy of antibody-virus molecular recognition
......Page 562
AFM tip chemistry
......Page 563
Protocol: Tip functionalization with antibodies
......Page 564
RNA release and single molecule unfolding
......Page 566
Protocol: Full genome studies of HRV1A
......Page 568
References......Page 570
High-Speed Atomic Force Microscopy Techniques for Observing Dynamic Biomolecular Processes......Page 574
Introduction......Page 575
Survey of Requirements for High-Speed Bio-AFM Imaging......Page 576
Bare mica surface......Page 577
Preparation of planar lipid bilayers......Page 579
Electrostatic immobilization......Page 582
Streptavidin 2D crystals as substrates......Page 583
Preparation of streptavidin 2D crystals......Page 584
Use of streptavidin 2D crystals as substrates......Page 585
Control of Diffusional Mobility......Page 587
Streptavidin 2D crystals......Page 588
p97 2D crystals......Page 590
Low-Invasive Imaging......Page 591
UV Flash-Photolysis of Caged Compounds......Page 592
Cantilever Tip......Page 594
References......Page 595
Nanopore Force Spectroscopy Tools for Analyzing Single Biomolecular Complexes
......Page 598
Introduction
......Page 599
The Nanopore Method
......Page 600
Theory of force-driven molecular rupture
......Page 604
Analysis of constant-force experiments
......Page 608
Transformation of rupture-voltage histograms into voltage dependence of lifetime
......Page 609
DNA Unzipping Kinetics Studied Using Nanopore Force Spectroscopy
......Page 610
Maximum-likelihood analysis of voltage-ramp data
......Page 613
Histogram transformation method
......Page 615
Temperature rescaling of unzipping data
......Page 617
Conclusions and Summary
......Page 618
References......Page 620
Analysis of Single Nucleic Acid Molecules with Protein Nanopores
......Page 623
Background: Analysis of Nucleic Acids with Nanopores
......Page 625
Structure of the αHL nanopore
......Page 626
Nucleic acid analysis with αHL nanopores
......Page 627
Homopolymeric strand analysis with protein nanopores
......Page 628
Individual base recognition by αHL nanopores
......Page 629
Control of DNA translocation through nanopores
......Page 631
Electrical Recording with Planar Lipid Bilayers
......Page 633
Electrical recording equipment
......Page 634
Preparation of electrodes
......Page 635
Chambers
......Page 637
Preparing bilayers
......Page 638
Bilayer stability
......Page 639
Measuring bilayer capacitance
......Page 640
Inserting pores and adding DNA
......Page 641
Nanopore storage
......Page 642
Measurements with nanopores
......Page 643
Nanopore stability
......Page 644
Short single-stranded DNA or RNA
......Page 645
Long single-stranded DNA
......Page 646
Short dsDNA preparation
......Page 647
Filtering and sampling
......Page 648
Acquisition protocols
......Page 649
Analysis of single DNA/RNA molecules
......Page 650
References......Page 651