Single Molecule Analysis: Methods and Protocols (Methods in Molecular Biology, 2694)

✍ Scribed by Iddo Heller (editor), David Dulin (editor), Erwin J.G. Peterman (editor)

• Publisher: Humana
• Year: 2023
• Tongue: English
• Leaves: 511
• Category: Library

✦ Synopsis

This third edition volume expands on the previous editions with new discussions on the latest techniques and developments in the field. The chapters in this book are organized into four parts, and cover topics such as optical tweezers; single-molecule fluorescence tools; atomic force microscopy; magnetic tweezers; applications to virus protein shells, unfolding of proteins, nucleic acids, motor proteins, in vivo and in vitro; and protocols to establish specific surface interactions and perform force calibration. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Cutting-edge and thorough, Single Molecule Analysis: Methods and Protocols, Third Edition is a valuable resource for all researchers who wantto learn more about this exciting and still expanding field.

Chapters 2, 7, 8, 9, 12, 18, and 19 are available open access under a Creative Commons Attribution 4.0 International License via link.springer.com.

✦ Table of Contents

Preface
Contents
Contributors
Part I: Optical Tweezers
Chapter 1: Introduction to Optical Tweezers: Background, System Designs, and Applications
1 Introduction
1.1 History of Optical Tweezers
1.2 Optical Tweezers in Biology
2 Principles of Optical Tweezers Techniques
2.1 Forces in an Optical Trap
2.2 Principles of Trap Calibration
3 Optical Tweezers Systems
3.1 The Optical Trap
3.2 Environment of the Trap
3.3 Position and Force Detection
3.4 Trap Steering
3.5 Environment of the Setup
4 Optical Tweezers Approaches for Single-Molecule Analysis
4.1 Measurement Modes
4.2 Biological Assays in Optical Tweezers
4.3 Combining Optical Tweezers with Fluorescence Microscopy
5 Commercial Optical Tweezers Systems
6 Concluding Remarks
7 Notes
References
Chapter 2: Quantifying ATP-Independent Nucleosome Chaperone Activity with Single-Molecule Methods
1 Introduction
2 Materials
2.1 Instruments, Reagents, and Buffers for Sample Preparation
2.2 Instruments and Buffers for Single-Molecule Experiments
3 Methods
3.1 FACT Protein and Isolated Domain Production
3.2 NHP6A Protein Expression and Purification
3.3 Combining Nucleosome Position Sequences and Handles to Form Templates
3.4 Reconstituting Nucleosome Arrays
3.5 Force Disruption (FD) Using Optical Tweezers
3.6 Quantifying Force Disruption Data
3.7 Evaluating the Free Energy and Transition Barrier
3.8 Characterizing the Effects of a Chaperone Protein
3.9 Quantifying Changes to the Energy Landscape
3.10 Confocal Imaging (CI) and Kymographs in Combined Instrument
3.11 Survival Probability (SP) Across Cycles of Disruption and Release
3.12 Comparisons
4 Notes
References
Chapter 3: Protein Tethering for Single-Molecule Force Spectroscopy
1 Introduction
2 Materials
2.1 Synthesis of dsDNA Handles
2.2 Protein-Anchor Coupling
2.3 Preparation of Antibody-Coated Beads
2.4 Immobilization of Protein and DNA Constructs on Antibody-Coated Beads
3 Methods
3.1 Synthesis of DNA Tethers with Single-Stranded Overhang
3.2 Protein-Anchor Coupling Via Cysteines
3.3 Protein-Anchor Coupling Via Enzymatic Reaction
3.4 Ligation of the Anchor-Protein-Anchor Construct to the Long Tethers
3.5 Bead Preparation and Testing in Optical Tweezers
4 Notes
References
Chapter 4: Insect Cell-Based Expression of Cytoskeletal Motor Proteins for Single-Molecule Studies
1 Introduction
2 Materials
2.1 Bacmid Generation
2.1.1 Bacmid Selection and Plate Preparation
2.1.2 Recombinant Bacmid Generation
2.2 Bacmid Purification
2.2.1 Bacmid Culture Preparation
2.2.2 Bacmid Culture Purification
2.3 Insect Cell Transfection, Baculovirus Generation, and Protein Expression
2.3.1 Insect Cell Growth and Maintenance
2.3.2 Insect Cell Transfection
2.3.3 Baculovirus Generation and Protein Expression
2.4 Protein Purification
2.4.1 Protein Binding Via FLAG Tag
2.4.2 Protein Elution
2.5 Single-Molecule Assay
2.5.1 Microtubule Polymerization
2.5.2 Microtubule Slide Preparation
2.5.3 Single-Molecule Fluorescent Imaging
2.5.4 Optical Trapping Assay
3 Methods
3.1 Bacmid Generation
3.1.1 Preparation of the Bacmid Selection Plate
3.1.2 Recombinant Bacmid Generation
3.2 Bacmid Purification
3.2.1 Bacmid Culture Preparation
3.2.2 Bacmid Culture Purification
3.3 Insect Cell Transfection, Baculovirus Generation, and Protein Expression
3.3.1 Insect Cell Growth and Maintenance
3.3.2 Insect Cell Transfection
3.3.3 Baculovirus Generation and Protein Expression
3.4 Protein Purification
3.4.1 Protein Binding Via FLAG Tag
3.4.2 Protein Elution
3.5 Single-Molecule Assay
3.5.1 Microtubule Polymerization
3.5.2 Microtubule Slide Preparation
3.5.3 Single-Molecule Fluorescent Imaging
3.5.4 Optical Trapping Assay
4 Notes
References
Chapter 5: Probing Mitotic Chromosome Mechanics Using Optical Tweezers
1 Introduction
2 Materials
2.1 Optical Tweezers Instrument Construction
2.1.1 Microfluidics
2.1.2 Dual Optical Trap
2.1.3 Calibration and Validation
2.2 Chromosome Isolation and Sample Preparation
2.3 Data Acquisition
3 Methods
3.1 Optical Tweezers Setup
3.1.1 Microfluidics
3.1.2 Dual Optical Trap
3.1.3 Validating the High-Force Regime
3.1.4 Force Calibration at High Laser Powers
3.2 Chromosome Isolation and Sample Preparation
3.2.1 Cell Culture Maintenance
3.2.2 Chromosome Isolation
3.2.3 Chromosome Purification and Sample Preparation
3.3 Data Acquisition
3.3.1 Preparation of the Microfluidic System
3.3.2 Experimental Workflow
4 Notes
References
Part II: Single-Molecule Fluorescence Microscopy
Chapter 6: A Brief Introduction to Single-Molecule Fluorescence Methods
1 Introduction
1.1 A Brief Introduction to Fluorescence Spectroscopy
1.2 A History of Single-Molecule Fluorescence Microscopy
2 Fluorophores
2.1 Important Properties of Fluorescent Molecules Used for Single-Molecule Methods
2.2 Important Characteristics of Fluorescent Labels
2.3 Fluorophores Used for Single-Molecule Research
3 Methods
3.1 Microscopic Detection of Single Fluorophores
3.1.1 Light Sources
3.1.2 Filters and Dichroic Mirrors
3.1.3 Detectors in Single-Molecule Fluorescence Microscopy
3.1.4 Microscope Objectives
3.1.5 The Resolution of a Fluorescence Microscope
3.2 Key Imaging Modalities in Fluorescence Microscopy
3.2.1 Confocal Fluorescence Microscopy
3.2.2 Wide-Field Epi-Fluorescence Microscopy
3.2.3 Total Internal Reflection Fluorescence Microscopy (TIRF)
3.2.4 Selective-Plane Illumination Microscopy (SPIM)
3.3 Availability of Instrumentation
4 Measurables
4.1 Counting the Number of Fluorescent Molecules Within a Diffraction-Limited Spot
4.2 Localization of Single Molecule
4.3 Detection of Motion of Single Molecules
4.4 Single-Molecule Localization Super-Resolution Microscopy
4.5 Colocalization of Fluorescent Molecules
4.6 Förster Resonance Energy Transfer (FRET)
4.7 Fluorescence Polarization
5 Concluding Remarks
References
Chapter 7: Single-Molecule Fluorescence Microscopy in Sensory Cilia of Living Caenorhabditis elegans
1 Introduction
2 Materials
2.1 Anesthetizing and Mounting C. elegans
2.2 Microscope Setup
3 Methods
3.1 Preparing Imaging Pads
3.2 Mounting C. elegans
3.3 Imaging
3.4 Data Analysis
3.4.1 Kymograph Analysis
3.4.2 Single-Particle Tracking
4 Notes
References
Chapter 8: Lattice Light-Sheet Motor-PAINT: A Method to Map the Orientations of Microtubules in Complex Three-Dimensional Arra...
1 Introduction
2 Materials
2.1 Protein Purification and Labeling
2.2 Sample Preparation
2.3 Microscope and Data Analysis Setup
3 Methods
3.1 Protein Purification and Labeling
3.2 Sample Preparation
3.3 Imaging
3.4 Analysis
4 Notes
References
Chapter 9: Fluorescence Microscopy of Nanochannel-Confined DNA
1 Introduction
2 Materials
2.1 Fabrication of Chips
2.2 Chemicals
2.3 Buffers
2.3.1 Buffer for DNA Experiments
2.3.2 Protocol for Preparing 1 L 5x TBE Buffer
2.3.3 Protocol for Staining 1 mL of 10 μg/mL DNA at a dye:bp Ratio of 1:10 in 0.5x TBE Buffer
2.3.4 Protocol for Preparing 400 μL Loading Buffer and 100 μL DNA in Loading Buffer
2.4 DNA Samples
2.5 Fluorescence Microscopy
2.6 Addressing the Chip
2.7 Data Analysis
3 Methods
3.1 Design and Fabrication of Chips
3.1.1 Design
3.1.2 Fabrication
3.1.3 Definition of Alignment Marks
3.1.4 Definition of Nanochannels
3.1.5 Definition of Microchannels (Fused Silica and Silicon-Borosilicate Glass)
3.1.6 Processing of Access Holes
3.1.7 Sealing of the Chips
3.2 Chemicals
3.2.1 Fluorescent Labeling of DNA
3.3 Running Experiments (Loading of DNA)
3.4 Data Analysis
4 Notes
References
Chapter 10: Single-Molecule FRET X
1 Introduction
2 Materials
2.1 Annealing the DNA Nanostructure
2.2 Single-Molecule Imaging
2.3 Data Acquisition and Analysis Software
3 Methods
3.1 Single-Molecule Fluorescence Microscopy, Data Acquisition, and Analysis
3.2 Single-Molecule FRET X
3.3 Single-Molecule FRET X to Probe Multiple Points of Interest in a Single Nanostructure
4 Notes
References
Chapter 11: Single-Molecule Fluorescence Imaging of DNA Replication Stalling at Sites of Nucleoprotein Complexes
1 Introduction
2 Materials
2.1 Surface Functionalization
2.2 Rolling-Circle Template Preparation
2.2.1 2-kb Rolling-Circle DNA Template
2.2.2 18-kb Rolling-Circle DNA Template
2.3 Experimental Setup
2.4 Visualization and Analysis of DNA Replication Stalling
3 Methods
3.1 Glass Coverslip Surface Functionalization
3.2 Rolling-Circle DNA Template Construction
3.2.1 2-kb Rolling-Circle DNA Template
3.2.2 18-kb Rolling-Circle DNA Template
3.3 Experimental Setup
3.4 Visualization and Analysis of DNA Replication Stalling
3.4.1 gRNA-647 Hybridization
3.4.2 dCas9-gRNA647 Pre-incubation
3.4.3 Reaction Scheme 1: Roadblock Pre-incubation Assay
3.4.4 Reaction Scheme 2: Roadblock in-Solution Assay
3.4.5 Data Quantification
4 Notes
References
Chapter 12: Measuring Transcription Dynamics of Individual Genes Inside Living Cells
1 Introduction
2 Materials
2.1 Plasmids and Plasmid Amplification (For Budding Yeast)
2.2 Plasmids and Plasmid Amplification (Mammalian Cells)
2.3 Yeast Culture and Loop Integration (for Budding Yeast)
2.4 Cell Culture and Loop Integration (Mammalian Cells)
2.5 Microscopy Sample Preparation (For Budding Yeast)
2.6 Microscopy Sample Preparation (Mammalian Cells)
2.7 Microscope (Both Yeast and Mammalian Cells)
3 Methods
3.1 Strategic Decisions Before Cloning and Integration of Stem Loops and Coat Protein (for Both Yeast and Mammalian Cells)
3.2 Creating Yeast Strain with Stem Loops
3.3 Coat Protein Integration (Yeast)
3.4 Creating Cell Line with Stem Loops (Mammalian)
3.5 Stable Coat Protein Integration (Mammalian)
3.6 Sample Preparation for Microscopy (yeast)
3.7 Sample Preparation for Microscopy (Mammalian)
3.8 Live Cell Microscopy
3.8.1 Widefield or Confocal
3.9 Data Analysis/Processing
4 Notes
References
Chapter 13: Single-Molecule FRET-Resolved Protein Dynamics - from Plasmid to Data in Six Steps
1 Introduction
1.1 smFRET Enables Monitoring of Protein Dynamics
1.2 Transformation of Plasmid into Competent Escherichia coli
1.3 Expression of the Protein of Interest
1.4 Purification of the Protein of Interest
1.5 Dye Labeling of the Protein of Interest
1.6 Detection of Protein Dynamics Using smFRET
1.7 Data Processing
2 Materials
2.1 Transformation of Plasmid into Competent E. coli
2.2 Expression of the Protein of Interest
2.3 Purification of the Protein of Interest
2.4 Fluorescent Labeling of the Protein of Interest
2.5 smFRET of Protein Dynamics
2.6 smFRET Data Processing
3 Methods
3.1 Transformation of Plasmid into Competent E. coli
3.2 Expression of the Protein of Interest
3.3 Purification of the Protein of Interest
3.3.1 His-Tag Purification
3.3.2 Anion Exchange Chromatography (AEX)
3.3.3 Size-Exclusion Chromatography (SEC)
3.4 Dye Labeling of the Protein
3.5 smFRET of Protein Dynamics
3.5.1 Coverslip Cleaning
3.5.2 Vectabond Coating
3.5.3 First PEG Coating
3.5.4 Second PEG Coating
3.5.5 smFRET Data Acquisition
3.6 smFRET Data Processing
4 Notes
5 Data Availability
References
Part III: Atomic Force Microscopy
Chapter 14: Atomic Force Microscopy: An Introduction
1 Introduction
2 Basics of AFM
2.1 Setup and Principle
2.1.1 Tip Sample Interactions
2.2 Operation Modes
2.2.1 Contact Mode
2.2.2 Oscillating Modes
2.2.3 Bimodal Imaging Mode
2.2.4 Jumping Mode Imaging
2.2.5 Force Spectroscopy
2.2.6 Operation Environments
3 High-Speed AFM and Its Recent Developments
4 AFM Applications in Biological Sciences
5 Conclusion
References
Chapter 15: Atomic Force Microscopy of Viruses: Stability, Disassembly, and Genome Release
1 Introduction
2 Materials
2.1 AFM Cantilevers
2.2 Particles Adsorption on the Surface of Solid Substrates
3 Methods
3.1 Imaging
3.2 Tip Dilation
3.3 Imaging Virions at Different Environment Conditions
3.4 Mechanical Properties of Protein Shells: Nanoindentation
3.5 Genome Externalization and Mechanical Properties of Cargo
3.6 Mechanical Fatigue, Disassembly, and Aging
3.7 Layer-by-Layer Disassembly with Mechanical Fatigue in a Multilayered Virus
3.8 AFM/Fluorescence Combination
4 Notes
References
Chapter 16: Unfolding and Refolding Proteins Using Single-Molecule AFM
1 Introduction
2 Materials
3 Methods
3.1 Cleaning of the Cover Slides
3.2 Gold and Nickel/Chromium Alloy Thermal Deposition
3.3 Polyprotein Expression and Purification
3.4 Activating Gold-Coated Cover Slides
3.5 Protein Deposition to the Cover Slide
3.6 Cantilever Alignment
3.7 Sample Fixation into the AFM/Piezo
3.8 Engaging the Protein Sample with the Cantilever Holder
3.9 Thermal Spectrum and Finding the Surface
3.10 Cantilever Spring Constant Value Determination
3.11 Protein Unfolding Using the Force Extension (FX) Mode
3.12 PID Adjustment for Force Clamp (FC) Measurements
3.13 Protein Unfolding Using the Force Clamp (FC) Mode
3.14 Refolding a Protein Using the FC Mode
4 Notes
References
Chapter 17: Visualizing Molecular Dynamics by High-Speed Atomic Force Microscopy
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents
3 Methods
3.1 Surface Preparation
3.2 Sample Stage Preparation
3.3 Preparation of Large Unilamellar Vesicles (LUVs) for a Lipid Bilayer Surface
3.4 Cantilever Mounting
3.5 Laser Alignment and Cantilever Oscillation
3.6 Sample Deposition and Incubation (See Fig. 2b)
3.7 Mount Sample Stage on Top of the Chamber
3.8 Approach
3.9 Imaging
3.10 Three Example Experiments with Specific Adaptions to the Protocol
3.10.1 Real-Time Formation of a 2D HIV Viral Capsid Protein (CA) Lattice on Mica (See Fig. 9a, b)
3.10.2 Seeded Growth of Self-Assembling Supramolecular Fibers on a Lipid Bilayer (See Fig. 9c)
3.10.3 Mode of Action of the Antibiotic Teixobactin (See Fig. 9d)
3.11 Image Processing
4 Notes
References
Part IV: Magnetic Tweezers and Other High-Throughput Methods
Chapter 18: An Introduction to Magnetic Tweezers
1 Brief History and Application of Magnetic Tweezers
2 Description of a Magnetic Tweezers Apparatus
2.1 Magnet Configuration
2.2 Illumination
2.3 Bead Position Tracking Algorithm
2.4 Temperature Control
2.5 Surface Functionalization and Nucleic Acid Construct Fabrication
3 Physical Principles
3.1 Force and Torque Origin
3.2 Force Calibration
3.3 Estimating the Spatiotemporal Resolution of Magnetic Tweezers
3.3.1 Tracking Resolution and Stability
3.3.2 Thermal Noise
3.4 Using Torque Spectroscopy in Magnetic Tweezers
4 Combining Magnetic Tweezers with Other Techniques
5 Applications of Magnetic Tweezers in Single-Molecule Biophysics
6 Perspectives
References
Chapter 19: Surface Functionalization, Nucleic Acid Tether Characterization, and Force Calibration for a Magnetic Tweezers Ass...
1 Introduction
2 Materials
2.1 General Materials
2.2 Nitrocellulose Passivation
2.3 Preparation of Small Unilamellar Vesicles (SUVs)
2.4 Lipid Bilayer Passivation
2.5 High-Throughput Magnetic Tweezers
3 Methods
3.1 Nitrocellulose Passivation
3.2 Preparation of Small Unilamellar Vesicles (SUVs)
3.3 Lipid Bilayer Passivation
3.4 Tether Selection
3.5 Force Calibration
4 Notes
References
Chapter 20: Correlated Single-Molecule Magnetic Tweezers and Fluorescence Measurements of DNA-Enzyme Interactions
1 Introduction
2 Materials
2.1 Setup
2.1.1 Controlled Focus Difference Between MT and Fluorescence
2.1.2 Light Source for MT
2.1.3 Different Magnet Configurations
2.1.4 Vibration Isolation
2.1.5 GPU Tracking for MT
2.1.6 Micromirrors to Generate TIRF Illumination
2.2 Flow Cells
2.3 Superparamagnetic Beads
2.4 Buffers
2.5 Oxygen Scavenger System
2.6 DNA Materials
2.7 Protein Materials
3 Methods
3.1 Alignment of the Setup
3.1.1 General Approach
3.1.2 Mounting a Laser
3.1.3 Aligning the Tweezers Illumination (TI)
3.1.4 The Detection Channel
3.1.5 Inserting and Aligning Tiny Mirrors (Tm1 & Tm2)
3.2 Antidigoxigenin-Coated Coverslips
3.2.1 Making Holes on Glass and Cutting Parafilm Channels
3.2.2 Cleaning the Coverslips
3.2.3 Preparing the 1% Polystyrene-Toluene Solution (10 mg/mL)
3.2.4 Coating the Coverslips Without Holes (Making the Glass Hydrophobic)
3.2.5 Assembling the Flow Cells
3.2.6 Preparing the Flow Cells for Measurements
3.3 PEGylated Coverslips
3.3.1 Cleaning the Coverslips
3.3.2 Amino-Silanization and PEGylation
3.3.3 Just Prior to the Measurement
3.4 Coating Carboxylated Beads with Antidigoxigenin
3.5 Buffers
3.6 Oxygen Scavenger
3.6.1 Protocol for 100 mM Trolox
3.6.2 Protocol for PCA
3.6.3 Protocol for PCD
3.7 DNA
3.8 Cloning the CRISPR Target into pUC19
3.8.1 Constructs for Correlated MT-TIRF Measurements
3.9 Fluorescently Labeled Protein
3.10 Measurements Combining Magnetic Tweezers and TIRF Microscopy
3.10.1 Microscope Setup
3.10.2 Flow Cell Preparation
3.11 R-Loop Formation Supercoiling Experiments (PEGylated Flow Cell)
3.11.1 Immobilization of Reference Beads
3.11.2 Preparation and Addition of Magnetic Beads with Tethered DNA
3.11.3 Initial Assessment of the Tethers
3.11.4 Identification of Single Supercoilable DNA Tethers
3.11.5 Force Calibration
3.11.6 Monitorization of R-Loop Formation and Dissociation with Simultaneous Fluorescence Imaging
3.12 1D Diffusion Measurements (Using an Antidigoxigenin-Coated Flow Cell)
3.12.1 Immobilization of Reference Beads
3.12.2 Preparation and Addition of Magnetic Beads with Tethered DNA
3.12.3 Initial Assessment of the Tethers
3.12.4 Diffusion Measurements
3.13 Data Analysis
3.13.1 Correlating MT Trajectories with Fluorescence Images
3.13.2 Generation of Kymographs
4 Notes
References
Chapter 21: Detecting DNA Loops Using Tethered Particle Motion
1 Introduction
2 Materials and Equipment
2.1 Microscope and Camera
2.2 Personal Computer
2.3 Particle Tracking Software
2.4 Buffers
2.5 Preparation of DNA Tethers
2.6 Flow Chambers
2.7 DNA Looping Protein
3 Methods
3.1 Flow Chambers
3.1.1 Slide and Coverslip Cleaning
3.1.2 Assembly
3.2 Tethering Particles with DNA
3.2.1 Amplify DNA with Biotin- and Digoxigenin Labels at Opposite Ends
3.2.2 Wash Beads
3.2.3 Coat Microchamber with Anti-digoxigenin
3.2.4 Attach DNA to Surface and Tether Beads
3.3 DIC Microscopy
3.4 Data Collection
3.5 Preprocessing Data
3.5.1 Drift Correction
3.5.2 Symmetry Test
3.6 Loop Detection
4 Results
5 Conclusion
6 Notes
References
Chapter 22: Single-Cell Measurements Using Acoustic Force Spectroscopy (AFS)
1 Introduction
2 Materials
2.1 AFS Experimental Setup
2.2 Surface Chemistry Buffers
3 Methods
3.1 Calibrating the Impedance of the AFS Chip
3.2 Preparation of the Surface
3.3 Measurements on Cells
3.4 Data Analysis
4 Notes
References
Chapter 23: DNA Origami-Based Single-Molecule Force Spectroscopy and Applications
1 Introduction
2 Materials
2.1 Design and Force Calculation of DNA Origami Force Clamps
2.2 Molecular Cloning of M13mp18 Phage Genomic DNA
2.3 Preparation of Doubly Labeled DNA Complement
2.4 Preparation of DNA Origami Force Clamps
2.5 Confocal Single-Molecule Fluorescence Measurements
2.6 Data Analysis
3 Methods
3.1 Design and Force Calculation of DNA Origami Force Clamps
3.2 Cloning of Custom DNA Sequences and Preparation of M13 Phage Genomic DNA
3.2.1 Molecular Cloning Using M13 Phage Vectors
3.2.2 Transformation
3.2.3 Clone Selection
3.2.4 Large-Scale Phage Production and Purification of DNA Scaffolds
3.3 Preparation of Fluorescently Labeled DNA Complementary to the DNA Origami Spring
3.4 Preparation of DNA Origami Force Clamps
3.4.1 Linearization of Scaffold DNA
3.4.2 Preparation of DNA Origami Folding Mix
3.4.3 Preparative Agarose Gel Electrophoresis
3.5 Confocal Single-Molecule Fluorescence Measurements
3.6 Data Analysis
4 Notes
References
Index