This new volume of Methods in Cell Biology looks at methods for analyzing of biophysical methods in cell biology. Chapters cover such topics as AFM, traction force microscopy, digital holographic microscopy, single molecule imaging, video force microscopy and 3D multicolor super-resolution screening
Covers sections on model systems and functional studies, imaging-based approaches and emerging studies
Chapters are written by experts in the field
Cutting-edge material

Contributors xv

Preface xxv

CHAPTER 1 Single-Molecule Imaging of Cytoplasmic Dynein In Vivo 1

Introduction 2

1. Visualization of Cytoplasmic Dynein In Vivo 2

2. Image Analysis 7

3. Conclusion 10

4. Methods 10

References 11

CHAPTER 2 Single-Molecule Imaging in Live Cell Using Gold Nanoparticles 13

Introduction and Rationale 14

1. Gold Nanoparticle Synthesis and Functionalization 15

2. Photothermal Imaging 20

3. Live Cell Imaging 22

Conclusion 26

Aclcnowledgments 26

References 26

CHAPTER 3 Quantitative Measurement of Transcription Dynamics in Living Cells 29

1. Visualizing Transcription in Living Cells 30

2. Experimental Protocols 32

3. Cell Segmentation 32

4. Measuring Spot Intensity 35

5. Correcting for Background MS2-GFP Level 35

6. Tracking Algorithms 37

7. Additional Cell Properties 39

8. Summary 39

References 40

CHAPTER 4 A Easy-to -Use Single-Molecule Speckle Microscopy Enabling Nanometer-Scale Flow and Wide-Range Lifetime Measurement of Cellular Actin Filaments 43

Introduction 44

1. Methods 46

2. Perspectives 56

Acknowledgrnents 57

References 57

CHAPTER 5 Dissecting Microtubule Structures by Laser Ablation 61

Introduction 62

1. Theoretical Framework 63

2. Microtubule Organization Measurements 68

Discussion and Conclusion 72

Acknowledgments 73

References 73

CHAPTER 6 Quantifying Mitochondrial Content in Living Cells 77

Introduction 78

1. Basic Protoco] (96-Well Glass Bottom Plate) 79

2. Alternate ProLocol (Cel!ASIC Microtluidic Flow Chamber) 86

3. Important Considerations for Successful MitoGraph

Performance 87

4. Beyond Wild-type Mitochondria in Buddi ng Yeast Imaged with Spinning-Disk Confocal Microscopy 89

Acknowledgments 93

Supplementary data 93

References 93

CHAPTER 7 High-Content 30 Multicolor Super-Resolution Localization Microscopy 95

Introduction 96

1. Sample Preparation . I02

2. Imaging Acquisition and Image Analysis 107

Conclusions and Outlook 112

Acknowledgments 1 13

References 113

CHAPTER 8 Superresolution Measurements In Vivo: Imaging Drosophila Embryo by Photoactivated Localization Microscopy 119

Introduction 120

1. Embryo Preparation 121

2. Sample Mounting 124

3. Optical Setup 125

4. Imaging 132

5. Data Analysis 133

6. Summary and Outlook 140

Acknowledgments 140

References 140

CHAPTER 9 Refractive Index Measurements of Single, Spherical Cells Using Digital Holographic Microscopy 143

Introduction 144

1. Setup 146

2. Measurement Preparation 148

3. Data Analysis 149

4. Discussion 154

5. Summary 156

References 157

CHAPTER 10 Construction, Imaging, and Analysis of FRET-Based Tension Sensors in Living Cells 161

Introduction 162

1. Design, Production, and Validation of Tension Sensors 163

2. Imaging of FRET-Based Biosensors 171

3. Methods of Analysis of FRET Images 176

4. Summary 183

References 184

CHAPTER 11 Single-Cell Mechanics: The Parallel Plates Technique 187

Introduction 188

1. Experimental Setup 189

2. Microplates 191

3. Cell Preparation 197

4. Experim ental Protocols 198

5. Discussion 206

Supplementary Data 208

References 209

CHAPTER 12 Atomic Force Microscopy-Based Force Measurements on Animal Cells and Tissues 211

Introduction 212

1. Experimental Setup 213

2. Sample Preparation 216

3. AFM and Optical Imaging 218

4. Measuring Cell and Tissue Stiffness 220

5. Measuring Adhesion 225

6. Further Applications 228

Conclusions 230

Acknow ledgments 231

References 231

CHAPTER 13 Measuring the Elasticity of Plant Cells with Atomic Force Microscopy 237

Introduction 238

1. Sample Preparation and Mounting 238

2. Atomic Force Microscopy 240

3. Experimental Design 243

4. Discussion 250

5. Notes 250

Acknowledgments 252

References 253

CHAPTER 14 Dual Pipette Aspiration: A Unique Tool for Studying lntercellular Adhesion 255

Introduction 256

1. Overview of the DPA Setup 257

2. Preparing the Pipettes 257

3. Preparing the Aspi.ration Assay 259

4. Cell Micromanipulation 261

5. Discussion 264

General Conclusions 265

References 266

CHAPTER 15 Measurement of Cell Traction Forces with lmageJ 269

Introduction 270

1. Force Measurement Principle 271

2. Critical Experimental Parameters 274

3. Critical Numerical Parameters 276

4. Preparation of Patterned Polyacrylamid e Gels with Fiducial Markers 280

5. Image Acquisition 283

6. Image Analysis, Estimation of Displacement, and Traction Force Fields 283

Conclusion 285

Supplementary Data 286

References 286

CHAPTER 16 Micropillar Substrates: A Tool for Studying Cell Mechanobiology 289

Introduction 290

1. Substrate Fabrication 292

2. Substrate Characterization 296

3. Substrate Functionalization 298

4. Cells Seeding and Imaging 301

5. Image Analysis and Evaluation of Traction Force 303

6. Discussion and Perspectives 305

Acknowledgments 306

References 306

CHAPTER 17 Mapping Forces and Kinematics during Collective Cell Migration 309

Introduction 310

1. Experimental Tools to Map Forces and Kinematics during Collective Cell Migration 311

2. Computational Tools to Map Forces and Kinematics during Collective Cell Migration 320

General Conclusions 328

Acknowledgments 328

References 328

CHAPTER 18 Practical Aspects of the Cellular Force Inference Toolkit (CellFIT) 331

Introduction 332

1. The Basic Steps in Ce!LFIT 334

2. Working with CellFIT Output 347

Acknowledgments 349

References 350

CHAPTER 19 Quantification of Collagen Contraction in Three-Dimensional Cell Culture 353

Introduction 354

1. Method 355

2. Pseudo-speck.le Microscopy 359

3. Software 360

4. Data Analysis 367

5. Discussion 369

General Conclusion 370

Acknowledgments 370

References 371

CHAPTER 20 Generation of Biocompatible Droplets for In Vivo and In Vitro Measurement of Cell-Generated Mechanical Stresses 373

Introduction 374

1. Methods 375

2. Discussion 387

Conclusion 388

References 389

CHAPTER 21 Laser Induced Wounding of the Plasma Membrane and Methods to Study the Repair Process 391

Introduction 392

1. Cell Deposition 393

2. Photodamage and Imaging 394

3. Following Plasma Membrane Damage and Repair 399

4. lrnage Analysis 401

5. Data Analysis 402

6. Discussion 405

General Conclusions 406

References 406

CHAPTER 22 Electrofusion of Giant Unilamellar Vesicles to Cells 409

Introduction 410

1. Preparation of GUYs by Electroforrnation 411

2. Electrofu sion of GUVs to Cells 414

3. Discussion 418

References 420

CHAPTER 23 Measurement and Manipulation of Cell Size Parameters in Fission Yeast 423

Introduction 424

1. Measurement of Size Parameters of Single Fission Yeast Cells 425

2. Microchannel Assay for Cell Diameter Manipulation 430

Conclusions 435

Acknowledgments 435

References 435

CHAPTER 24 Methods for Rectifying Cell Motions In Vitro: Breaking Symmetry Using Microfabrication and Microfluidics 437

Introduction 438

Relevance of Cell Migration In viva 438

Origin of Symmetry Breaking In viva and the Need for Controlled In vitro Approaches: Microfabrication and Microfluidics 439

1 Breaking Symmetry with Topography : Fabrication of a Topographical Pattern 439

2. Breaking Symmetry with Chemical Gradient: Preparation of the Fibronectin Gradient. 444

3. Cell Migration Experiments 447

4. Discussion 450

Conclusions 451

References 451

CHAPTER 25 Analyzing Bacterial Movements on Surfaces 453

Introduction 454

1. Preparing Bacterial Suspension 455

2. Tracking Bacteria on Solid Surfaces 457

3. Tracking Bacteria on Cells 464

4. Discussion Points 467

Conclusions 468

References 468

CHAPTER 26 Advances in Single-Cell Experimental Design Made Possible by Automated Imaging Platforms with Feedback Through Segmentation 471

Introduction 472

1. In vitro Experiments where Automation is lmportant. 473

2. Preparation of Cells Described in this C1\CHAPTER 475

3. Automation Methods 476

4. Discussion 483

5. Outlook 485

Ack nowledgments 487

References 487

Volumes in Series 489

Index 501

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