Optical Tweezers: Methods and Protocols

Preface
Contents
Contributors
Part I: Historic Views on the Invention of Lasers and Optical Tweezers
	Chapter 1: The Invention of the Laser
		1 The Story
		2 Postscript
		3 A Brief Statement on My Sources and Research
	2: Art Ashkin and the Origins of Optical Trapping and Particle Manipulation
		1 Early Predictions of Optical Forces
		2 Ashkin´s Earliest Work with Radiation Pressure
		3 The Terrestrial Affairs of Optical Forces
		4 The Advent of the Single-Beam Optical Trap
		5 The Impact of Optical Traps on Biophysics and the Life Sciences
		6 Concluding Remarks
		References
Part II: Technical Advances
	3: Synthesis of Germanium Nanospheres as High-Precision Optical Tweezers Probes
		1 Introduction
		2 Materials
			2.1 Materials for Germanium Nanosphere Synthesis
			2.2 Materials for Lipid-Bilayer Coating of Germanium Nanospheres
		3 Methods
			3.1 Synthesis of GeNTOPs
			3.2 Lipid-Bilayer Coating of Germanium Spheres
		4 Notes
		References
	Chapter 4: Angular Optical Trapping to Directly Measure DNA Torsional Mechanics
		1 Introduction
			1.1 Glossary of Mathematical Symbols
		2 Materials
			2.1 General Components of an AOT
			2.2 Cylinder Fabrication
			2.3 DNA Sample and Chamber Preparation
		3 Methods
			3.1 Cylinder Fabrication and Coating
				3.1.1 Fabrication Protocol
				3.1.2 Coating Protocol
			3.2 Sample Preparations
				3.2.1 Torsionally Constrained (TC) DNA Sample Preparation
				3.2.2 DNA Sample Chamber Preparation
			3.3 DNA Extension and Force Measurement
				3.3.1 Detect the Axial Cylinder Displacement zcyl
				3.3.2 Find the Surface: htrap = 0
				3.3.3 Determine the Focal Shift Ratio
				3.3.4 Calibrate Axial Position Detector and Axial Trap Stiffness
				3.3.5 Force and Its Trap Height Dependence
			3.4 Input Polarization Control and Characterization
				3.4.1 Polarization State Control
				3.4.2 Detection of the Polarization State of the Laser Trap
			3.5 Torque Generation and Detection Principle
			3.6 Torque Detector Calibration
				3.6.1 Torque Detector Sensitivity Calibration
				3.6.2 Angular Trap Stiffness Calibration
			3.7 The Constant-Extension Method and Its Applications
		4 Notes
		References
	Chapter 5: Implementation of 3D Multi-Color Fluorescence Microscopy in a Quadruple Trap Optical Tweezers System
		1 Introduction
		2 Materials
			2.1 Quadruple-Trap Optical Tweezers
				2.1.1 Optical Trap Steering and Force Detection Unit
				2.1.2 Microfluidics
				2.1.3 Custom-Written LabVIEW Software
			2.2 Epi Fluorescence Excitation
				2.2.1 Laser Combiner
				2.2.2 Beam Expander and Diffuser
			2.3 Fluorescence Detection
			2.4 Bright-Field Imaging
			2.5 Buffer Solutions
				2.5.1 Bleach Cleaning Solutions (See Subheading 3.4.1)
				2.5.2 Solutions for Passivation of the Flow Cell (See Subheading 3.4.2) (See Note 8)
				2.5.3 Buffer Solutions for the Assays (See Subheadings 3.5 and 3.6)
		3 Methods
			3.1 Fluorescence Excitation Design and Alignment
				3.1.1 Choice of Beam Expansion
				3.1.2 Choice of Lasers
				3.1.3 Alignment of Fluorescence Excitation Path
			3.2 Fluorescence Detection Design and Alignment
				3.2.1 Choice of Camera
				3.2.2 Calculation of Imaging Relay Lenses (L9 and L10, Fig. 1)
				3.2.3 Choice of Dichroic Mirrors and Filters
				3.2.4 Alignment of Fluorescence Detection Path
			3.3 Design of Z-Stack Imaging Module
				3.3.1 Z-Stage Alignment and Implementation
			3.4 Assay Preparation
				3.4.1 Bleach Cleaning
				3.4.2 Flow Cell Passivation
				3.4.3 Starting a Dual Trap Experiment
				3.4.4 Starting a Quadruple Trap Experiment
				3.4.5 Turning on Fluorescence
			3.5 3D Imaging of Multiple DNA Molecules Between Two Beads
				3.5.1 Catching Beads, DNA, and Force Calibration
				3.5.2 3D Imaging of Multiple DNA Molecules Between Two Beads
				3.5.3 Data Analysis
			3.6 Dual-Color Imaging of Intercalator Binding to dsDNA
				3.6.1 Catching DNA Between the Bead Pairs
				3.6.2 Wrapping the Two DNA Strand Around One Another
				3.6.3 Dual-Color Imaging of Intercalator Binding to Two dsDNA Molecules
				3.6.4 Data Analysis
		4 Notes
		References
	Chapter 6: One-Dimensional STED Microscopy in Optical Tweezers
		1 Introduction
		2 Materials
			2.1 Dual-Trap Optical Tweezers with Confocal Fluorescence and STED Microscopy
				2.1.1 Optical Tweezers System
				2.1.2 Confocal Fluorescence and STED Imaging
			2.2 Gold Nanoparticle (Au-NP) and Fluorescent Bead Samples
		3 Methods
			3.1 Course Alignment
				3.1.1 Course Alignment STED or Excitation Beams
				3.1.2 Forward Alignment  APD
				3.1.3 Backward Alignment  APD
				3.1.4 Alignment  PMT
			3.2 Prepare Au Nanoparticle Sample
			3.3 Focal-Intensity Distribution Imaging and Fine Alignment
				3.3.1 Focal-Intensity Distribution Imaging
				3.3.2 Fine Co-alignment of Excitation, STED, and APD Spots
			3.4 Correction of Misalignment and Aberrations
				3.4.1 Correcting Coma-Like Aberrations
				3.4.2 Correcting a Tilted Focal-Intensity Distribution
				3.4.3 Correcting Spherical Aberrations
			3.5 Alignment of STED Phase Plate
				3.5.1 Adjusting Phase Plate Rotation
				3.5.2 Adjusting Phase Plate Position
			3.6 Verifying Resolution Enhancement by 1D-STED
		4 Notes
		References
	Chapter 7: Temperature Quantification and Temperature Control in Optical Tweezers
		1 Heat Generation in Optical Tweezers: Causes and Consequences
			1.1 Temperature Generation in Optical Systems
				1.1.1 Laser-Induced Objective Heating
				1.1.2 Temperature Changes of Optical and Optomechanical Components
				1.1.3 Convective Airflow Around the Optical System
			1.2 Temperature of the Sample
				1.2.1 Bulk Temperature of the Sample
				1.2.2 Local Temperature at the Focus of the Trapping Beam
			1.3 Experimental Consequences of Heat Generation in the Sample
				1.3.1 Systematic Errors in Trap Calibration
				1.3.2 Unwanted Convective Flow
				1.3.3 Temperature Effects on Biomolecular Systems
			1.4 Minimizing Temperature Effects in an Optical Tweezers Setup
				1.4.1 Tools and Strategies at Hand to Suppress Thermal Effects
				1.4.2 Instrumentation Design for Temperature Control and Modulation
		2 Materials
			2.1 Optical Tweezer with Back Focal-Plane Interferometry
				2.1.1 Optical Tweezers with Force Detection
				2.1.2 Temperature Control at the Objective-Condenser System
		3 Methods
			3.1 Calibrating Optical Traps
				3.1.1 Passive Calibration
				3.1.2 Active Calibration
			3.2 Preparing the Nano-positioning Stage for Oscillations
			3.3 Preparing the Oscillatory Voltage Waveform
			3.4 Preparing the Flow Chamber
			3.5 Executing the Stage Oscillations While Acquiring the Particle´s Position Fluctuation
			3.6 Data Analysis to Calibrate the Trap Stiffness
			3.7 Temperature Quantification Via Active Calibration
			3.8 Operating the Temperature-Control Collars
		4 Notes
		References
	Chapter 8: High-Resolution Optical Tweezers Combined with Multicolor Single-Molecule Microscopy
		1 Introduction
		2 Materials
			2.1 High-Resolution Optical Tweezers Setup
			2.2 Multicolor Fluorescence Confocal Microscope
			2.3 Brightfield Imaging System
			2.4 Trap AOM RF Synthesis
			2.5 Data Acquisition and Instrument Control System
			2.6 Optical Tweezers and Confocal Microscope Alignment
			2.7 Sample Chamber Assembly
			2.8 Functionalization of Beads
			2.9 DNA Tether Constructs
			2.10 Oxygen Scavenging System/Fluorescence Imaging Buffer
			2.11 Preparation of Beads and Sample for Labeled GQ Construct Experiment
		3 Methods
			3.1 Setup of RF Synthesizer for Trapping Laser AOM
			3.2 Setup of the High-Resolution Optical Tweezers Module
			3.3 Setup of Multicolor Fluorescence Confocal Microscope Module: Excitation Laser Path
			3.4 Setup of Multicolor Fluorescence Confocal Microscope Module: Emission Path
			3.5 Adjustment of the Front Objective Correction Collar
			3.6 Calibration of Trap and Confocal Spot Positioning
			3.7 Preparation of DNA Tether Constructs
				3.7.1 Preparation of GQ-containing DNA Tether Construct
				3.7.2 Preparation of QD-probe Cryptic Binding Hairpin Construct
			3.8 Preparation of Oxygen Scavenging and Antiblinking Solutions
			3.9 Bead Functionalization
			3.10 Assembly of Laminar Flow Sample Chambers
			3.11 Preparation of Beads Directly Prior to Tweezers Experiment
			3.12 Sample Buffer Preparation for GQ Experiment
			3.13 Setting Up the Instrument for an Experiment
			3.14 Confirming Instrument Operation with Simultaneous Tweezers and Fluorescence Measurements Using G-Quadruplex-Containing Te...
		4 Notes
		References
Part III: DNA, DNA Motors and DNA-Protein Interactions
	Chapter 9: Generating Negatively Supercoiled DNA Using Dual-Trap Optical Tweezers
		1 Introduction
			1.1 Principle of Optical DNA Supercoiling
			1.2 Experimental Considerations
		2 Materials
			2.1 DNA Construct Preparation (See Also Fig. 1a)
			2.2 DNA Purification
			2.3 Core Components for Dual-Trap Optical Tweezers Set-up (Fig. 3)
			2.4 Microfluidic Flow System (Fig. 4)
			2.5 Cleaning of Microfluidic Flow System
			2.6 Passivation of Flow Cell and Tubing
			2.7 Reagents for Single-Molecule Experiments
			2.8 General Materials
			2.9 Data Acquisition
		3 Methods
			3.1 Generation of Biotin-Labeled End-Capped  DNA
				3.1.1 Phosphorylation of  DNA
				3.1.2 Ligation of Phosphorylated DNA Segments
				3.1.3 DNA Purification
			3.2 Optical Trapping Instrumentation
			3.3 Microfluidic Flow System
				3.3.1 Checking for Blockages in the Flow System
				3.3.2 Cleaning the Flow System
				3.3.3 Passivation
				3.3.4 ODS Sample Preparation
			3.4 Tethering DNA Between Optically Trapped Beads
			3.5 Testing the DNA Integrity
			3.6 Performing an ODS Experiment
				3.6.1 Generating Underwound DNA Using Type 1 End-Closed Torsionally Constrained DNA Substrates
				3.6.2 Generating Underwound DNA Using Type 2 End-Closed Torsionally Constrained DNA Substrates
				3.6.3 Generating Underwound DNA Using Type 3 End-Closed Torsionally Constrained DNA Substrates
				3.6.4 Generating a Fixed Reduction in Lk when Starting with Torsionally Unconstrained End-Closed  DNA
			3.7 Testing the Temporal Stability of the Reduced Lk
			3.8 Calibrating the Fixed Change in Lk
		4 Notes
		References
	Chapter 10: Force-Activated DNA Substrates for In Situ Generation of ssDNA and Designed ssDNA/dsDNA Structures in an Optical-T...
		1 Introduction
		2 Materials
			2.1 Designing Force-Activated DNA Substrates
				2.1.1 A 1-kbp Segment of Exactly 50% GC
				2.1.2 A 20-bp Hairpin with 33-nt Helicase Binding Site
				2.1.3 Extending to Longer Hairpins and Higher GC Content
				2.1.4 120-bp Hairpins with Distinct Runs of Different GC Content
			2.2 Assembling DNA Templates for Final PCR Amplification
				2.2.1 Initial PCR Amplification of DNA Handles
				2.2.2 Preparing the Substrate for Ligation Using Restriction Enzymes
				2.2.3 Cloning Substrate into E. coli for Efficiency and Accuracy
			2.3 Final PCR Amplification with Labeled Primers
			2.4 Site-Specific Nicking of the Force-Activatable Substrate
			2.5 Coupling Substrates to Coverslips Through Covalent Click Chemistry
			2.6 Using the Constructs in an Optical-Trapping Assay
		3 Methods
			3.1 Designing Force-Activated DNA Substrates
				3.1.1 A 1-kbp Segment of Exactly 50% GC
				3.1.2 A 20-bp Hairpin with 33-nt Helicase Binding Site
				3.1.3 Extending to Longer Hairpins and Higher GC Content
				3.1.4 120-bp Hairpins with Distinct Runs of Different GC Content
			3.2 Assembling DNA Templates for Final PCR Amplification
				3.2.1 Initial PCR Amplification of DNA Handles
				3.2.2 Preparing the Substrate for Ligation Using Restriction Enzymes
				3.2.3 Cloning Substrate into E. coli for Efficiency and Accuracy
			3.3 Final PCR Amplification with Labeled Primers
			3.4 Site-Specific Nicking of the Force-Activatable Substrate
			3.5 Coupling Substrates to Coverslips Through Covalent Click Chemistry
			3.6 Using the Constructs in an Optical-Trapping Assay
		4 Notes
		References
	Chapter 11: Probing the Interaction Between Chromatin and Chromatin-Associated Complexes with Optical Tweezers
		1 Introduction
		2 Materials
			2.1 Construction of Nucleosome Array Substrates
				2.1.1 Linearization of Nucleosome Array Templates
				2.1.2 Ligation of Nucleosome Array Template to Biotinylated Adapters
				2.1.3 Purification of Mouse Mammary Tumor Virus (MMTV)  DNA
				2.1.4 Reconstitution of Nucleosome Arrays
			2.2 Evaluation of Nucleosome Arrays
				2.2.1 Magnesium-Induced Precipitation
				2.2.2 Micrococcal Nuclease (MNase) Digestion
				2.2.3 ScaI Digestion
			2.3 Optical Tweezers Assay
			2.4 Single-Molecule Data Analysis
		3 Methods
			3.1 Construction of Nucleosome Array Substrates
				3.1.1 Linearization of Nucleosome Array Templates
				3.1.2 Ligation of Nucleosome Array Template to Biotinylated Adapters
				3.1.3 Purification of MMTV  DNA
				3.1.4 Reconstitution of Nucleosome Arrays
			3.2 Evaluation of Nucleosome Arrays
				3.2.1 Magnesium-Induced Precipitation
				3.2.2 MNase Digestion
				3.2.3 ScaI Digestion
			3.3 Optical Tweezers Assay
				3.3.1 Sample and Instrument Preparation
				3.3.2 Recording Force-Extension Trajectories of Single Chromatin Tethers
			3.4 Single-Molecule Data Analysis
				3.4.1 Force-Extension Analysis
				3.4.2 Cluster Assignment
		4 Notes
		References
	Chapter 12: Simultaneous Mechanical and Fluorescence Detection of Helicase-Catalyzed DNA Unwinding
		1 Introduction
		2 Materials
			2.1 DNA Template Preparation
			2.2 Protein and Buffer Preparation
				2.2.1 BLM Helicase
				2.2.2 RPA-eGFP
			2.3 Combined Optical Tweezers and Fluorescence Microscope
		3 Methods
			3.1 DNA Template Preparation
				3.1.1 N-DNA Template Construction
				3.1.2 T-DNA Template Construction
			3.2 Protein Purification
				3.2.1 BLM Expression and Purification
				3.2.2 RPA-eGFP Expression and Purification
			3.3 Single-Molecule DNA Unwinding Assay
				3.3.1 BLM-Catalyzed DNA Unwinding in SYTOX Reaction Buffer
				3.3.2 BLM-Catalyzed Unwinding in the Presence of RPA-eGFP
				3.3.3 BLM-Catalyzed DNA Unwinding in the Presence of SYTOX and RPA-eGFP
			3.4 Data Analysis
			3.5 RPA Activates BLM´s Bidirectional Unwinding from a  Nick
		4 Notes
		References
	Chapter 13: CRISPR/Cas9 On- and Off-Target Activity Using Correlative Force and Fluorescence Single-Molecule Microscopy
		1 Introduction
		2 Materials
			2.1 Purification of Labeled crRNA Guides
			2.2 In-Vitro Transcription of tracrRNA
			2.3 Biotinylation of λ-DNA
			2.4 Preparation of Trolox Solution
			2.5 Preparation of PCA/PCD Oxygen Scavenger
			2.6 Complexing the Cas9 Protein and Guide  RNAs
			2.7 Cleaning and Passivation of the Microfluidics
			2.8 Finding the Correct Objective and Condenser z-Position
			2.9 Optically Trapping Beads and Catching  DNA
			2.10 Performing wtCas9 On-Target Cutting Experiments
		3 Methods
			3.1 Purification of Labeled crRNA Guides
			3.2 In-Vitro Transcription of tracrRNA
			3.3 Biotinylation of λ-DNA
			3.4 Preparation of Trolox Solution
			3.5 Preparation of the PCA/PCD Oxygen Scavenger System
			3.6 Complexing the Cas9 Protein and Guide  RNAs
			3.7 Cleaning and Passivation of the Sample Syringes, Microfluidics Flow Cell and Tubing
			3.8 Finding the Correct Objective and Condenser z-Position
			3.9 Optically Trapping Beads and Catching DNA Molecules Using the Microfluidics
			3.10 Imaging dCas9 Binding
				3.10.1 Imaging dCas9 On-Target Binding
				3.10.2 Imaging Force Induced Off-Target Binding
			3.11 Analysis of Cas9 Binding Events
				3.11.1 Analysis of Off-Target Binding Events by Intensity Time Binning
				3.11.2 Analysis of Off-Target Binding Event by Trajectory Detection
			3.12 Performing wtCas9 On-Target Cutting Experiments
		4 Notes
		References
Part IV: Protein (Un)Folding
	Chapter 14: Cotemporal Single-Molecule Force and Fluorescence Measurements to Determine the Mechanism of Ribosome Translocation
		1 Introduction
		2 Materials
			2.1 Preparation of DNA Handles
			2.2 Preparation of Stalled Ribosome Complexes
				2.2.1 Preparation of mRNA Template
				2.2.2 Preparation of Deacylated tRNAs
				2.2.3 Charging Deacylated tRNAs for Ribosome Stalling
				2.2.4 Preparation of Ribosome Initiation Complexes on the Template mRNA
				2.2.5 Preparation of TM-Val Translation Stalling Mix
				2.2.6 Preparation of Stalled Ribosomes and Annealing to the 5′ DNA Handle
				2.2.7 Preparation of Translation Factor Mix
				2.2.8 Preparation of Oxygen Scavenging and Triplet State Quenching Solutions
				2.2.9 Preparation of 0.1% Streptavidin Coated Polystyrene Bead Solution
			2.3 Microfluidic Chamber Construction
			2.4 Single Molecule Force and Fluorescence Assay to Measure Real-Time Translation
		3 Methods
			3.1 Preparation of DNA Handles
			3.2 Preparation of Stalled Ribosome Complexes
				3.2.1 Preparation of mRNA Template
				3.2.2 Preparation of Deacylated tRNAs
				3.2.3 Charging Deacylated tRNAs for Ribosome Stalling
				3.2.4 Preparation of Ribosome Initiation Complexes on the Template mRNA
				3.2.5 Preparation of TM-Val Translation Stalling Mix
				3.2.6 Preparation of Stalled Ribosomes and Annealing to the 5′ DNA Handle
				3.2.7 Preparation of Translation Factor Mix
				3.2.8 Preparation of Oxygen Scavenging and Triplet State Quenching Solutions
				3.2.9 Preparation of 0.1% Streptavidin Coated Polystyrene Bead Solution
			3.3 Microfluidic Chamber Construction
			3.4 Single-Molecule Force and Fluorescence Assay to Measure Real-Time Translation
				3.4.1 Sample Preparation for the Different Channels of the Microfluidic Chamber
				3.4.2 Forming Tethers and Confirming Stalled Complexes
				3.4.3 Restarting Translation by Flowing in Translation Mix
			3.5 Data Analysis
		4 Notes
		References
	Chapter 15: Using Single-Molecule Optical Tweezers to Study the Conformational Cycle of the Hsp90 Molecular Chaperone
		1 Introduction
		2 Materials
			2.1 Expression and Purification of Hsp90 from Saccharomyces cerevisiae
			2.2 Dimerization Via Cysteine-Stabilized Coiled-Coil and ssDNA Maleimide Oligo Attachment
			2.3 Dimerization Via YBBR-Tag and ssDNA Maleimide Oligo Attachment
			2.4 Preparation of DNA Handles
			2.5 Functionalization of Beads
			2.6 Optical Tweezers Measurements
		3 Methods
			3.1 Expression and Purification of Hsp90 from Saccharomyces cerevisiae
				3.1.1 Plasmid Transformation into Competent Cells and Sequencing
				3.1.2 Protein Expression and Purification
			3.2 Dimerization Via Cysteine-Stabilized Coiled-Coil and ssDNA Maleimide Oligo Attachment
			3.3 Dimerization Via YBBR-Tag and ssDNA Maleimide Oligo Attachment
			3.4 Measuring Hsp90 in the Optical Tweezers
				3.4.1 General Protocol for Measuring a Protein Construct in the Dual-Beam Optical Tweezers
				3.4.2 Hsp90 Monomer-Based Unfolding and Refolding Studies
				3.4.3 Hsp90 Charged Linker Characterization: An Example of Equilibrium Measurements
				3.4.4 Hsp90 Middle-Domain Attachment Dimer: An Example Application of Jump Experiments
			3.5 Preparation of DNA Handles
			3.6 Functionalization of Beads
			3.7 Conclusions and Outlook
		4 Notes
		References
	Chapter 16: Tethering Complex Proteins and Protein Complexes for Optical Tweezers Experiments
		1 Introduction
			1.1 Enzymatic Tethering
			1.2 Autocatalytic Tethering
		2 Materials
			2.1 Expression and Purification of Sfp and BirA
			2.2 Attachment of CoA Moiety to Oligos
			2.3 Biotinylation of Avi-Tagged Proteins with BirA
			2.4 CoA-Oligo Reaction with Protein of Interest with Sfp
			2.5 Generation of DNA Coupled Beads
			2.6 Protein Immobilization and Tethering
			2.7 Expression/Purification of SpyCatcher
			2.8 Coupling of SC to Oligonucleotides
			2.9 Generation of Labeled Molecular Handles and Ligation to SC-Oligonucleotides
			2.10 Tethering SpyTagged Proteins in Optical Tweezers Experiments
			2.11 Equipment
		3 Methods
			3.1 Enzymatic Tethering
				3.1.1 Design and Purification of Target Proteins
				3.1.2 Expression and Purification of Modifying Enzymes
				3.1.3 Preparation of Coenzyme A-Oligonucleotide Adducts
				3.1.4 Protein Modification
				3.1.5 Bead-DNA Coupling
				3.1.6 DNA Handle Synthesis
				3.1.7 Protein Immobilization
				3.1.8 Tether Assembly
			3.2 Autocatalytic Tethering
				3.2.1 Target Protein Design and Purification
				3.2.2 SpyCatcher Purification
				3.2.3 SpyCatcher-DNA Coupling
				3.2.4 DNA Handle Synthesis
				3.2.5 SpyCatcher-DNA Handle Coupling
				3.2.6 Protein-DNA Coupling
				3.2.7 Tether Assembly
		4 Notes
		References
	Chapter 17: Single-Molecule Manipulation Study of Chaperoned SNARE Folding and Assembly with Optical Tweezers
		1 Introduction
		2 Materials
			2.1 Preparation of SNARE Constructs
				2.1.1 Protein Expression
				2.1.2 Protein Purification
				2.1.3 Protein Biotinylation and Cleavage
			2.2 DNA Handles
			2.3 Crosslinking
			2.4 Optical Tweezers Assay
				2.4.1 Home-Built Dual-Trap High-Resolution Optical Tweezers
				2.4.2 Home-Built Flow Control System
				2.4.3 Microfluidic Flow Chamber Preparation
				2.4.4 Single-Molecule Experiments with Optical Tweezers
		3 Methods
			3.1 Preparations Before Execution of Single-Molecule Assays
				3.1.1 Protein Constructs and the Experimental Setup
				3.1.2 Plasmid Preparation
				3.1.3 Protein Expression
				3.1.4 Protein Purification
				3.1.5 Biotinylation
				3.1.6 SNARE Complex Formation and DNA Handle Crosslinking
				3.1.7 Preparation of the Microfluidic Flow Chamber
				3.1.8 Installation and Alignment of the Microfluidic Chamber
			3.2 Optical Tweezers Assays for Neuronal SNARE Construct
				3.2.1 Preparations of the Flow Control System
				3.2.2 Measure the Force-Extension Curves of the SNARE Complex in the Presence of Munc18-1
				3.2.3 Measure Reversible SNARE Folding/Assembly Transitions at Constant Trap Separations
			3.3 Data Analysis
		4 Notes
		References
	Chapter 18: Using Optical Tweezers to Monitor Allosteric Signals Through Changes in Folding Energy Landscapes
		1 Introduction
		2 Materials
			2.1 Transformation of E. coli and Expression of PKA Regulatory Subunit (PKA-R) Constructs
			2.2 cAMP-Coupled Agarose Resin Preparation
			2.3 cAMP-Coupled Agarose Resin-Based Protein Purification of PKA-R Constructs
			2.4 Preparation of Thiol-Modified Double-Stranded Oligonucleotide (dsOligo) for the Covalent Attachment of dsDNA Handles to PK...
			2.5 DTDP (2,2′-Dithiodipyridine) Activation of Cysteine-Modified PKA-R Constructs and Covalent Attachment of dsOligos
			2.6 Functional Selection of the Chimeric Protein-dsOligo Constructs (See Note 3)
			2.7 Preparation of dsDNA Handles (See Note 4)
			2.8 Ligation of Chimeric Protein-dsOligo Constructs with the dsDNA Handles
			2.9 Optical Tweezers
		3 Methods
			3.1 Transformation of E. coli and Expression of the PKA Regulatory Subunit (PKA-R) Constructs (Fig. 2c)
			3.2 cAMP-Coupled Agarose Resin Preparation
			3.3 cAMP-Coupled Agarose Resin-Based Protein Purification of PKA-R Constructs
			3.4 Preparation of Thiol-Modified dsOligo (CS1 and CS2) for DNA Handle Attachment to PKA-R Constructs
			3.5 DTDP Activation of Cysteine-Modified PKA-R Constructs and Formation of the Chimeric Protein-dsOligo
			3.6 Functional Selection of the HPH (Chimeric Protein-dsOligo) Constructs
			3.7 Preparation of dsDNA for Ligation of Handles to HPH
			3.8 Ligation of Chimeric Protein-dsOligo Constructs with the dsDNA Handles
			3.9 Optical Tweezers Experiments
			3.10 Analysis of Unfolding and Refolding Transitions Observed in Optical Tweezers Trajectories
			3.11 The Effects of Binning and the Number of Data Points on Fitted Energy Landscape Parameters
			3.12 Using Bootstrapping to Estimate the Errors in the Folded and Unfolded-State Lifetimes and Distances to the Transition Sta...
			3.13 Dissecting the Effect of cAMP Binding and Interdomain Interactions in PKA-R
		4 Notes
		References
Part V: Cytoskeletal Motors and Proteins
	Chapter 19: High-Speed Optical Traps Address Dynamics of Processive and Non-Processive Molecular Motors
		1 Introduction
			1.1 Background
			1.2 Ultrafast Optical Trapping
		2 Materials
			2.1 Beads Functionalization
				2.1.1 Neutravidin Beads
					Biotinylated Latex Beads
					Neutravidin-Coated Fluorescent Latex Beads
				2.1.2 N-Ethylmaleimide (NEM) Modified Beads
				2.1.3 α-Actinin-Coated Fluorescent Beads
					Purification of HaloTagged, Actin-Binding Domain of α-Actinin (HT-ABD)
					Coupling HT-ABD to Beads
					Fluorescent Labeling of Beads
			2.2 Silica Beads
			2.3 Rhodamine F-Actin & Biotinylated Actin
				2.3.1 F-Actin
				2.3.2 Biotinylated F-Actin
			2.4 Flow Cell
			2.5 Myosin-V Experiments
			2.6 Myosin-II Experiments
			2.7 Optical Trap Instrument (AOD Configuration)
				2.7.1 Trapping Control Unit
				2.7.2 Imaging Unit
				2.7.3 Detection Unit
			2.8 Optical Trap Instrument (EOD Configuration)
		3 Methods
			3.1 Trapping Beads Functionalization
				3.1.1 Neutravidin-Coated Beads
					Biotinylated Latex Beads
					Neutravidin Fluorescent Beads
				3.1.2 Preparation of N-Ethylmaleimide Modified Myosin Beads
				3.1.3 α-Actinin Coated Fluorescent Beads
					Purification of Halo-Tagged, Actin-Binding Domain of α-Actinin (HT-ABD)
					Bead-Actin Linkages: Coupling of HT-ABD to Beads
					Labeling of HT-ABD Beads
			3.2 Preparation of Silica Beads
			3.3 Fluorescent and Biotinylated F-Actin
				3.3.1 F-Actin
				3.3.2 Biotinylated F-Actin
			3.4 Flow Cell Preparation
			3.5 Optical Trap Alignment (AODs)
				3.5.1 AOD Alignment
				3.5.2 EOD Alignment
			3.6 System Calibration (Table 1)
				3.6.1 Calibration of the nm-Stabilization System (z and x-y Stabilization)
				3.6.2 Trap Position (MHz to nm or V to nm)
				3.6.3 Power and Stiffness (MHz vs. W), QPD (MHz vs. pN/nm)
			3.7 Sample Preparation
				3.7.1 Processive Myosin V
				3.7.2 Non-Processive Myosin II
			3.8 Measurement Protocol
			3.9 Data Analysis
				3.9.1 Event Detection
				3.9.2 Duration Analysis
				3.9.3 Analysis of the Motor Protein Working Stroke
				3.9.4 Quantification of the Ensemble Averages
				3.9.5 Analysis of Processive Runs
				3.9.6 Run Length Correction for Pushing Forces
		4 Notes
		References
	Chapter 20: Microtubule Dumbbells to Assess the Effect of Force Geometry on Single Kinesin Motors
		1 Introduction
		2 Materials
			2.1 Casein Solution
			2.2 Microtubule Preparation
			2.3 Coating of Microspheres with Anti-His Antibodies and Kinesin for the Single-Bead Assay
			2.4 Glucose Oxidase and Catalase (POC) Oxygen Scavenger Preparation
			2.5 Nanopatterning of Rectangular Ridges
			2.6 Silica Sphere Suspension for Use as Pedestals in the Three-Bead Assay
			2.7 Chamber Preparation for the Optical Tweezers Assay
				2.7.1 Beads and Material for the Single-Bead Assay
				2.7.2 Beads and Material for the Three-Bead Assay
		3 Methods
			3.1 Casein Solution Preparation
			3.2 Microtubule Preparation
			3.3 Coating Microspheres with Anti-6xHis Antibodies and Kinesin for the Single-Bead Assay
			3.4 Glucose Oxidase and Catalase (GOC) Oxygen Scavenger Preparation
			3.5 Nanopatterning of Rectangular Ridges
			3.6 Preparation of Silica Sphere Suspension To Be Used as Pedestals in the Three-Bead Assay
			3.7 Chamber Preparation for the Optical Tweezers Assay
				3.7.1 Single-Bead Assay
				3.7.2 Three-Bead Assay
		4 Notes
		References
	Chapter 21: Single-Molecule Studies on the Motion and Force Generation of the Kinesin-3 Motor KIF1A
		1 Introduction
		2 Materials
			2.1 Construct Generation
			2.2 Mutagenesis
			2.3 E. coli Growth for Protein Expression
			2.4 Protein Purification
			2.5 MT-Binding and -Release Assay
			2.6 TIRF Assay
			2.7 Optical Tweezers Assay
		3 Methods
			3.1 KIF1A Construct Generation
			3.2 Mutagenesis
			3.3 E. coli Growth for Protein Expression
			3.4 Protein Purification
			3.5 MT-Binding and -Release Assay
				3.5.1 Preparation of  MTs
				3.5.2 MT-Binding and -Release Assay
			3.6 TIRF Assay
				3.6.1 Coverslip Cleaning
				3.6.2 Microscope Slide Assembly
				3.6.3 MT Preparation
				3.6.4 Slide Preparation
				3.6.5 TIRF Imaging
			3.7 Optical Tweezers Assay
				3.7.1 Coating of Polystyrene Microspheres
				3.7.2 Labeling of α-Casein with Sulfo-NHS-Biotin for MT Immobilization
				3.7.3 Coverslip Cleaning and Slide Assembly
				3.7.4 MT Preparation
				3.7.5 Slide Preparation
				3.7.6 Optical Tweezers Assay
		4 Notes
		References
	Chapter 22: Ultrafast Force-Clamp Spectroscopy of Microtubule-Binding Proteins
		Abbreviations
		1 Introduction
		2 Materials
			2.1 Laser-Tweezers Instrument for UFFC Assay
				2.1.1 Microscope and Other Parts
				2.1.2 Stage Stabilization
				2.1.3 UFFC Feedback Regimes
				2.1.4 Data Transfer Layout
			2.2 Pedestal Bead Immobilization
			2.3 Pedestal Bead Functionalization
				2.3.1 Functionalization of Pedestals Using SNAP-GBP
				2.3.2 Coating Pedestals with Biotinylated Anti-GFP Antibodies
				2.3.3 Recruitment of GFP-Tagged MAPs
			2.4 Microtubule Dumbbell Preparation
				2.4.1 Preparation of MTs
				2.4.2 Preparation of Dumbbell Beads Coated with Anti-Tubulin Antibodies
				2.4.3 Preparation of Dumbbell Beads Coated With Anti-DIG-Antibodies
			2.5 Ultrafast Force-Clamp Assay
				2.5.1 Preparation of Chamber with Functionalized Pedestals and MT
		3 Methods
			3.1 Laser-Tweezers Instrument for UFFC Assay
				3.1.1 Microscope and Other Paths (Fig. 2b)
				3.1.2 Stage Stabilization
				3.1.3 UFFC Feedback Regimes
				3.1.4 Data Transfer Layout
			3.2 Pedestal Bead Immobilization
				3.2.1 Partial-Melting Method
				3.2.2 Adsorption Method
			3.3 Testing the Strength of Pedestal Immobilization
				3.3.1 Monitoring Thermal Vibration (Standard Deviation (SD) Method)
				3.3.2 Pulling Pedestal Beads via Optical Trap
			3.4 Pedestal Bead Functionalization
				3.4.1 Functionalization of Pedestals Using SNAP-GBP
				3.4.2 Coating Pedestals with Biotinylated Anti-GFP Antibodies
				3.4.3 Recruitment of GFP-Tagged MAPs
			3.5 Microtubule Dumbbell Preparation
				3.5.1 Preparation of Microtubules
				3.5.2 Preparation of Dumbbell Beads Coated with Anti-Tubulin Antibodies
				3.5.3 Preparation of Dumbbell Beads Coated with Anti-DIG Antibodies
			3.6 Ultrafast Force-Clamp Assay
				3.6.1 Preparation of a Chamber with Functionalized Pedestals and Stabilized MTs
				3.6.2 Formation of MT Dumbbell
				3.6.3 Stretching of MT Dumbbell
				3.6.4 Determining the Contact Point Between MT Dumbbell and MAP-Coated Pedestal
				3.6.5 Verification of Pedestal Stability
				3.6.6 Ultrafast Force-Clamp Measurements
				3.6.7 Data Analysis
		4 Notes
		References
Part VI: Function and Mechanical Properties of Filaments
	Chapter 23: Catching the Conformational Wave: Measuring the Working Strokes of Protofilaments as They Curl Outward from Disass...
		1 Introduction
		2 Materials
			2.1 Instrument Design and Optical Layout
			2.2 Tubulin Purification
			2.3 Biotin-Tubulin Microtubule Seed Preparation
			2.4 Anti-His Bead Preparation
			2.5 Disposable Channel Slides
			2.6 Preparing Coverslip-Anchored Microtubules with Side-Bound Microbeads
			2.7 Measuring Pulses of Motion Generated by Curling Protofilaments
		3 Methods
			3.1 Tubulin Purification
			3.2 Biotin-Tubulin Microtubule Seed Preparation
			3.3 Anti-His Bead Preparation
			3.4 Preparing Disposable Channel Slides
			3.5 Preparing Coverslip-Anchored Microtubules with Side-Bound Microbeads
			3.6 Measuring Pulses of Motion Generated by Curling Protofilaments
			3.7 Analyzing Wave Assay Data
		4 Notes
		References
	24: Mechanics of Single Vimentin Intermediate Filaments Under Load
		1 Introduction
		2 Materials
			2.1 Vimentin Labeling
				2.1.1 Fluorescent Labeling
				2.1.2 Biotin Labeling
			2.2 Vimentin Filament Assembly
			2.3 Maleimide-Functionalized Beads
			2.4 Measurement
		3 Methods
			3.1 Vimentin Purification
			3.2 Vimentin Labeling
				3.2.1 Fluorescent Labeling
				3.2.2 Biotin Labeling
			3.3 Vimentin Filament Assembly
			3.4 Preparation of Maleimide-Functionalized Beads
			3.5 Measurement
		4 Analysis and Modeling
			4.1 Analysis of the Separate Regimes of Vimentin Filament Force-Strain Curves
			4.2 Analysis of Force-Clamp Measurements
			4.3 Modeling of Vimentin Filaments under Load
				4.3.1 Molecular Dynamics Simulations of Vimentin Dimers and Tetramers
				4.3.2 Two-State Model
				4.3.3 Three-State Model
				4.3.4 Monte-Carlo Simulations of Stretched Vimentin Filaments
				4.3.5 Modifications to Monte-Carlo Simulations
		5 Notes
		6 Variables and Parameters (Table 1)
		References
	25: Quantifying the Interaction Strength Between Biopolymers
		1 Introduction
		2 Materials
			2.1 Microtubule Sample Preparation
			2.2 Vimentin Filament Sample Preparation
			2.3 Bead Preparation
			2.4 General Measurement Preparation
		3 Methods
			3.1 Tubulin Purification and Labeling
			3.2 Microtubule Sample Preparation
			3.3 General Measurement Protocol
		4 Data Analysis-Extracting the Interaction Strength
			4.1 Classification of the Interaction Strength
			4.2 Geometry Factor
			4.3 Force Histograms
			4.4 Binding Rate
		5 Modeling-Two State Model of Single Interactions
			5.1 Rate Equations
			5.2 Force Rate
			5.3 Solution of Rate Equations
				5.3.1 Simulation of Interaction Events
				5.3.2 Numerical Solution
			5.4 Comparison with Experimental Data
			5.5 Derivation of the Energy Landscape
		6 Notes
		7 Variables and Parameters (Table 1)
		References
Part VII: Mechanosensing of Membrane Channels
	Chapter 26: Measuring αβ T-Cell Receptor-Mediated Mechanosensing Using Optical Tweezers Combined with Fluorescence Imaging
		1 Introduction
		2 Materials
			2.1 Generation of BW5147 Cell Lines
			2.2 T-Cell Activation IL-2 ELISA
			2.3 Media for Cell Culture and Single Cell Experimentation
			2.4 Refolding and Biotinylation of the pMHC VSV8/H-2Kb
			2.5 Bead Decoration with pMHC Molecules
			2.6 Chemical Etching of Coverslips and Flow Cell Assembly
			2.7 Determination of pMHC Coating Density
			2.8 Single Cell Activation Requirement Assay Preparation
			2.9 Anti-Digoxigenin Beads
			2.10 3500 Base-Pair DNA Linker
			2.11 Half Anti-Biotin Cleavage and Coupling to DNA Linker
			2.12 Single Molecule on Single Cell Assay Preparation
			2.13 Combined Optical Tweezers and Fluorescence Instrumentation
		3 Methods
			3.1 Generation of BW5147 Cell Lines
			3.2 T-Cell Activation IL-2 ELISA
			3.3 Refolding and Biotinylation of the pMHC VSV8/H-2Kb
			3.4 Bead Decoration with pMHC Molecules for Single Cell Activation Assay
			3.5 Chemical Etching of Coverslips
			3.6 Flow Cell Construction
			3.7 Determination of pMHC Coating Density
				3.7.1 Determination of pMHC Concentration at Limiting Molecular Count
				3.7.2 Determination of pMHC Concentration at High Molecular Count
			3.8 Single Cell Activation Requirement Assay Preparation
			3.9 Single Cell Activation Measurement
			3.10 Anti-Digoxigenin Beads
			3.11 Creation of DNA Linker with Half Anti-Biotin
				3.11.1 PCR Amplification of 3500 Base-Pair DNA Linker
				3.11.2 Antibody Splitting into Half Anti-Biotin
				3.11.3 Coupling of Half Anti-Biotin to 3500 bp DNA Linker
			3.12 Single Molecule on Single Cell Assay Preparation
			3.13 Single Molecule on Isolated Single Cell Measurement
		4 Notes
		References
Index