Biophysical Methods in Cell Biology (eBook)

Ewa Paluch (Herausgeber)

eBook Download: PDF | EPUB

2015 | 1. Auflage
568 Seiten
Elsevier Science (Verlag)
978-0-12-801326-7 (ISBN)

Infos

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

Inhalt

Front Cover 1
Methods in Cell Biology 2
Series Editors 3
Methods in Cell Biology 
4 
Copyright 
5 
Contents 6
Contributors 16
Preface 26
1. Single-molecule imaging of cytoplasmic dynein in vivo 28
Introduction 29
1. Visualization of Cytoplasmic Dynein In Vivo 29
1.1 Background 29
1.2 Experiment 30
1.2.1 Preparation of fission yeast zygotes 30
1.2.2 Observation of dyneins in the cytoplasm 31
1.2.3 Observation of dyneins on the microtubule 32
1.2.4 Discussion 32
1.2.4.1 Prebleaching to observe dyneins with higher SNR 32
1.2.4.1 Prebleaching to observe dyneins with higher SNR 32
1.2.4.2 Estimation of penetration depth of HILO 33
1.2.4.2 Estimation of penetration depth of HILO 33
2. Image Analysis 34
2.1 Tracking of Single Molecules 34
2.2 Confirmation of Single-Molecule Imaging 35
2.3 Analysis of Dynein Movement 36
3. Conclusion 37
4. Methods 37
4.1 Cell Culture 37
4.2 Preparation of Samples for Imaging 37
4.3 Microscopy 37
References 38
2. Single-molecule imaging in live cell using gold nanoparticles 40
Introduction and Rationale 41
1. Gold Nanoparticle Synthesis and Functionalization 42
1.1 Materials 42
1.1.1 List of chemicals required for the nanoparticle synthesis and functionalization 42
1.2 Nanoparticle Synthesis 43
1.3 Nanoparticle Functionalization with Nanobodies 45
1.4 Sample Characterization 46
1.4.1 Absorption spectra 46
1.4.2 Transmission electron microscope 46
1.4.3 Agarose gel electrophoresis 46
2. Photothermal Imaging 47
2.1 Materials 47
2.2 Principle 47
2.3 Experimental Setup 48
2.4 Resolution and Sensitivity 49
2.4.1 Resolution 49
2.4.2 Sensitivity 49
3. Live Cell Imaging 49
3.1 Cell surface labeling 49
3.2 2D Single-Particle Tracking 51
3.3 Particle Internalization 52
Conclusion 53
Acknowledgments 53
References 53
3. Quantitative measurement of transcription dynamics in living cells 56
1. Visualizing Transcription in Living Cells 57
2. Experimental Protocols 59
3. Cell Segmentation 59
4. Measuring Spot Intensity 62
5. Correcting for Background MS2-GFP Level 62
6. Tracking Algorithms 64
7. Additional Cell Properties 66
8. Summary 66
References 67
4. An easy-to-use single-molecule speckle microscopy enabling nanometer-scale flow and wide-range lifetime measurement of cell ... 70
Introduction 71
1. Methods 73
1.1 Preparation of Actin Probes 73
1.1.1 Required materials 74
1.1.2 Procedure for DyLight NHS ester labeling of actin 74
1.2 Electroporation Method for Delivery of DL-Actin to XTC Cells 75
1.2.1 Required materials 76
1.2.2 Procedure for electroporation of DL-actin into XTC cells 77
1.3 SiMS Imaging 78
1.3.1 Required materials 78
1.3.2 Examples of microscopy setups 79
1.3.3 Procedure for the SiMS imaging of DL-actin loaded XTC cells 79
1.4 Data Analysis 80
1.4.1 Nanometer-scale displacement measurement 81
1.4.2 Simultaneous analysis of actin dynamics with diverse timescales 81
2. Perspectives 83
Acknowledgments 84
References 84
5. Dissecting microtubule structures by laser ablation 88
Introduction 89
1. Theoretical Framework 90
2. Microtubule Organization Measurements 95
2.1 Extract and Sample Preparation 95
2.2 Microscopy and Laser Ablation Setup 95
2.3 Data Acquisition and Analysis 95
Discussion and Conclusion 99
Acknowledgments 100
References 100
6. Quantifying mitochondrial content in living cells 104
Introduction 105
1. Basic Protocol (96-Well Glass Bottom Plate) 106
1.1 Sample Preparation 106
1.1.1 Materials 106
1.1.2 Yeast culturing 107
1.1.3 96-well glass plate preparation and cell plating 108
1.2 Image Acquisition 108
1.2.1 3D spinning-disk confocal microscope setup 108
1.2.2 Imaging 108
1.3 Image Processing 109
1.3.1 Data preparation 109
1.3.2 Running data through MitoGraph software 110
1.3.3 Interpreting the data 111
1.3.3.1 Group files (one per folder) 111
1.3.3.2 Individual files (one for each TIFF file in the folder) 112
2. Alternate Protocol (CellASIC Microfluidic Flow Chamber) 113
2.1 Materials 113
2.2 Yeast Culturing Step 113
2.3 Cell Loading 113
3. Important Considerations for Successful MitoGraph Performance 114
3.1 Validation: Reproducibility and Accuracy 114
3.2 Optimal Magnification 114
3.3 Signal versus Background Noise Intensity Requirements 115
3.4 Effects of Spherical Aberration 115
3.5 Pros and Cons of “Surface Volume” and “Skeleton Volume” 116
4. Beyond Wild-type Mitochondria in Budding Yeast Imaged with Spinning-Disk Confocal Microscopy 116
4.1 Other Microscopy Modalities 116
4.1.1 Epifluorescence microscope 116
4.1.2 Laser-scanning confocal microscope 117
4.2 Mitochondrial Morphology Mutant Cells 117
4.3 Mitochondrial Networks in Nonyeast Cells 119
Acknowledgments 120
Supplementary data 120
References 120
7. High-content 3D multicolor super-resolution localization microscopy 122
Introduction 123
The Basis of an SMLM-Imaging Experiment 126
Hybridizing SMLM with High-Content Imaging 128
1. Sample Preparation 129
1.1 Equipment 129
1.2 Materials 129
1.3 Method 130
1.3.1 Cleaning slides and coverslips 130
1.3.2 Labeling with primary antibodies 131
1.3.3 Seeding the cells 132
1.3.4 Immunofluorescence 132
1.3.5 Coverslip mounting 133
2. Imaging Acquisition and Image Analysis 134
2.1 Equipment 134
2.2 Software 135
2.3 Method 135
2.3.1 Acquisition 135
2.3.2 Single-particle detection and reconstruction 137
2.4 Important considerations 137
2.4.1 Detectors used for the acquisition 137
2.4.2 Single-molecule detection and localization 138
2.4.3 SR drift correction and chromatic realignment 139
2.4.4 SR estimation and image reconstruction 139
Conclusions and Outlook 139
Acknowledgments 140
References 140
8. Superresolution measurements in vivo: Imaging Drosophila embryo by photoactivated localization microscopy 146
Introduction 147
1. Embryo Preparation 148
1.1 Materials 148
1.2 Embryo Staging and Dechorionation 149
1.3 Fixation 149
1.4 Devitellinization 151
2. Sample Mounting 151
2.1 Materials 151
2.2 Sample Mounting for High-Resolution Imaging 151
2.2.1 Coverslip preparation 151
2.2.2 Sample mounting 152
2.3 Single-Molecule Sample Preparation 152
3. Optical Setup 152
3.1 Principles of PALM 152
3.1.1 Photoactivable fluorescent protein 152
3.1.2 Principles of PALM 153
3.2 Basic Optical Setup 153
3.2.1 Illumination 153
3.2.2 Optical components 155
3.2.3 Optical schemes 155
3.2.4 3-D detection 155
3.3 Custom Optimization 157
3.3.1 Optimization the illumination 157
3.3.2 Synchronization 157
3.3.3 Stabilization 159
4. Imaging 159
5. Data Analysis 160
5.1 Single-Molecule Detection 160
5.2 Single-Molecule Localization 161
5.3 Drift Characterization and Correction 163
5.4 Estimation of PALM Localization Precision 163
5.5 PALM Data Visualization 165
5.6 Single-Molecule Counting 166
5.6.1 Characterization and correction for photoblinking 166
5.6.2 Estimation of labeling fraction 166
6. Summary and Outlook 167
Acknowledgments 167
References 167
9. Refractive index measurements of single, spherical cells using digital holographic microscopy 170
Introduction 171
1. Setup 173
2. Measurement Preparation 175
2.1 Materials 175
2.2 Method 175
2.3 Comments 176
3. Data Analysis 176
3.1 Determination of the Phase Distribution 177
3.2 Determination of the RI 179
4. Discussion 181
4.1 Setup Alterations 181
4.2 Assumption of Spherical Shape for Refractive Index Determination 181
4.3 Analysis Alterations 182
5. Summary 183
References 184
10. Construction, imaging, and analysis of FRET-based tension sensors in living cells 188
Introduction 189
1. Design, Production, and Validation of Tension Sensors 190
1.1 Required Constructs 190
1.2 Materials for Tension Sensor Creation 192
1.3 Design and Production of Constructs 193
1.3.1 Restriction enzyme-based cloning 193
1.3.1.1 Methods 193
1.3.2 Megaprimer-based overlap extension 195
1.3.2.1 Methods 195
1.3.3 Gibson assembly 196
1.3.3.1 Methods 197
1.4 Biochemically Validating a Tension Sensor 197
2. Imaging of FRET-Based Biosensors 198
2.1 Key Concepts of FRET Imaging 198
2.2 Materials and Equipment 199
2.2.1 Reagents 199
2.2.2 Equipment 200
2.3 Preparation for and Imaging of FRET-Based Tension Sensors 200
2.3.1 Experimental sample preparation 200
2.3.1.1 Methods 201
2.3.2 Detection of common imaging artifacts 201
2.3.2.1 Methods 201
2.3.3 Imaging of tension sensors and control constructs 202
2.3.3.1 Methods 203
3. Methods of Analysis of FRET Images 203
3.1 Quantification and Correction of Common Imaging Artifacts 203
3.1.1 Methods 205
3.1.2 Results 208
3.2 Examples of Common Errors in FRET Imaging 209
4. Summary 210
References 211
11. Single-cell mechanics: The parallel plates technique 214
Introduction 215
1. Experimental Setup 216
2. Microplates 218
2.1 Microplate Fabrication 219
2.1.1 Materials 219
2.1.2 Equipment 219
2.1.3 Method 219
2.2 Microplate Calibration 220
2.2.1 Materials 221
2.2.2 Method 221
2.3 Microplate Cleaning 221
2.3.1 Materials 221
2.3.2 Equipment 222
2.3.3 Method 222
2.4 Microplate Coating 223
2.4.1 Materials 223
2.4.2 Equipment 223
2.4.3 Method 223
2.4.4 Possible modifications for different coatings 223
3. Cell Preparation 224
3.1 Materials 224
3.2 Equipment 224
3.3 Method 224
4. Experimental Protocols 225
4.1 Cell Capture 225
4.2 Calibration of the Optical Sensor 226
4.3 Single-Cell Traction Force Measurements 226
4.3.1 Simple traction 226
4.3.2 Real-time single-cell response to stiffness 227
4.4 Single-Cell Rheology 227
4.4.1 Dynamic mechanical analysis 227
4.4.2 Creep experiment 232
4.4.3 Relaxation experiment 232
5. Discussion 233
Supplementary Data 235
References 236
12. Atomic force microscopy-based force measurements on animal cells and tissues 238
Introduction 239
1. Experimental Setup 240
1.1 Setup Design 240
1.2 Cantilever Calibration 241
2. Sample Preparation 243
2.1 Animal Pretreatment 243
2.2 Preparation of Measurement Buffers 244
2.3 Sample Immobilization 244
3. AFM and Optical Imaging 245
4. Measuring Cell and Tissue Stiffness 247
4.1 Important Parameters for Indentation Measurements 249
4.2 Analysis of Indentation Experiments 250
5. Measuring Adhesion 252
5.1 Chemical Force Microscopy 253
5.2 Single-Molecule Force Spectroscopy 254
5.3 Single-Cell Force Spectroscopy 255
6. Further Applications 255
Conclusions 257
Acknowledgments 258
References 258
13. Measuring the elasticity of plant cells with atomic force microscopy 264
Introduction 265
1. Sample preparation and mounting 265
1.1 Materials 266
1.2 Method 266
2. Atomic force microscopy 267
2.1 Material 267
2.2 Method 267
2.2.1 Generalized method 267
3. Experimental design 270
4. Discussion 277
5. Notes 277
Acknowledgments 279
References 280
14. Dual pipette aspiration: A unique tool for studying intercellular adhesion 282
Introduction 283
1. Overview of the DPA Setup 284
2. Preparing the Pipettes 284
2.1 Materials 284
2.2 Equipment 284
2.3 Methods 286
3. Preparing the Aspiration Assay 286
3.1 Materials 287
3.2 Equipment 287
3.3 Methods 287
4. Cell Micromanipulation 288
4.1 Materials 288
4.2 Equipment 288
4.3 Methods 288
5. Discussion 291
General Conclusions 292
References 293
15. Measurement of cell traction forces with ImageJ 296
Introduction 297
1. Force Measurement Principle 298
2. Critical Experimental Parameters 301
3. Critical Numerical Parameters 303
4. Preparation of Patterned Polyacrylamide Gels with Fiducial Markers 307
4.1 Materials 307
4.2 Equipment 307
4.3 Coating of 24×24mm Glass Coverslips with ECM 307
4.4 Silanization of 20×20mm Glass Coverslips 309
4.5 Polymerization of Polyacrylamide 309
5. Image Acquisition 310
6. Image Analysis, Estimation of Displacement, and Traction Force Fields 310
6.1 Preparation of ImageJ Software 310
6.2 Generation of the Parameter File 310
6.3 Measurement of the Cell Traction Energy 311
6.3.1 Displacement and force vectors 312
6.3.2 Mechanical energy stored in gel deformation 312
Conclusion 312
Supplementary Data 313
References 313
16. Micropillar substrates: A tool for studying cell mechanobiology 316
Introduction 317
1. Substrate Fabrication 319
1.1 Materials 320
1.2 Equipment 321
1.3 Methods 321
1.3.1 Fabrication of silicon wafer 321
1.3.2 Silanization of silicon wafer 322
1.3.3 Preparation of micropillar substrates 323
2. Substrate Characterization 323
3. Substrate Functionalization 325
3.1 Materials 325
3.2 Equipment 327
3.3 Methods 327
3.3.1 Preparation of Cy5.5 conjugated fibronectin 327
3.3.2 Microcontact printing of fibronectin 327
3.3.3 Adapted method for high-aspect ratio micropillars 328
4. Cells Seeding and Imaging 328
4.1 Materials 329
4.2 Equipment 329
4.3 Methods 329
5. Image Analysis and Evaluation of Traction Force 330
6. Discussion and Perspectives 332
Acknowledgments 333
References 333
17. Mapping forces and kinematics during collective cell migration 336
Introduction 337
1. Experimental Tools to Map Forces and Kinematics during Collective Cell Migration 338
1.1 Polyacrylamide Gel Preparation 339
1.1.1 Equipment and reagents 339
1.1.2 Protocol 339
1.1.2.1 Glass-bottom petri dish activation 339
1.1.2.2 Preparation of polyacrylamide gels 339
1.1.2.3 Gel functionalization 340
1.1.2.4 Collagen coating 340
1.2 Direct Cell Seeding 340
1.3 PDMS Membrane Barrier Assay 340
1.3.1 Reagents and materials 342
1.3.2 Clean room equipment 342
1.3.3 Protocol 343
1.3.3.1 SU-8 spinning on glass slides 343
1.3.3.2 SU-8 photolithography 343
1.3.3.3 SU-8 development and silanization 343
1.3.3.4 Fabrication of PDMS membranes 343
1.3.3.5 Membrane passivation and cell seeding 343
1.3.3.6 Membrane release 344
1.4 Magnetic PDMS Barrier Assay 344
1.4.1 Reagents and materials 344
1.4.2 Equipment 346
1.4.3 Protocol 346
1.4.3.1 Designing and printing the mold for the magnetic PDMS with the 3D printer 346
1.4.3.2 Fabrication of the magnetic PDMS stencil 346
1.4.3.3 Magnetic PDMS stencil sterilization, passivation, and cell seeding 346
2. Computational Tools to Map Forces and Kinematics during Collective Cell Migration 347
2.1 Image Acquisition 347
2.2 Image Registration 347
2.3 Computing Cell Velocities (PIV) 348
2.4 Computing Cell Forces 350
2.4.1 Computation of gel displacements at the interface with the cell monolayer 350
2.4.2 Computation of cell tractions at the interface with the cell monolayer 352
2.4.2.1 Boussinesq algorithm for infinite gel substrate of finite thickness 352
2.4.2.2 Finite element method (FEM) for gel substrate of finite thickness 353
2.4.3 Computation of inter- and intra-cellular stresses: MSM 353
2.4.3.1 Formulation of the problem 353
2.4.3.2 Solution of the problem 354
General Conclusions 355
Acknowledgments 355
References 355
18. Practical aspects of the cellular force inference toolkit (CellFIT) 358
Introduction 359
1. The Basic Steps in CellFIT 361
1.1 Image Segmentation 361
1.2 Mesh Generation 364
1.3 Angle Determination 365
1.4 Curvature Determination 367
1.5 Construct Young Equations 367
1.6 Assemble, Constrain, and Solve Tension Equations 369
1.7 Construct Laplace Equations 370
1.8 Assemble, Constrain, and Solve Pressure Equations 370
1.9 Display Results 371
1.10 Evaluate Solutions 373
2. Working with CellFIT Output 374
Acknowledgments 376
References 377
19. Quantification of collagen contraction in three-dimensional cell culture 380
Introduction 381
1. Method 382
1.1 Sample Preparation 382
1.1.1 Materials 384
1.1.1.1 Cell culture 384
1.1.1.2 Multicellular cancer cells spheroids 384
1.1.1.3 3D collagen assay 384
1.1.2 Method 384
1.1.2.1 Cell culturing 384
1.1.2.2 Multicellular cancer cells spheroids 384
1.1.2.3 3D collagen assay 385
2. Pseudo-speckle Microscopy 386
2.1 Microscopy 386
2.2 Basic Image Corrections 387
3. Software 387
3.1 General Parameters (Figure 3, Panel III.A) 388
3.2 Parameters for Edge Detection (Figure 3, Panel III.B) 388
3.3 Parameters for Cross-Correlation (Figure 3, Panel III.C) 390
3.4 Interpolation and Filter Parameters (Figure 3, Panel III.D) 392
3.5 Data and Image Recording and Data Structure 392
4. Data Analysis 394
5. Discussion 396
General Conclusion 397
Acknowledgments 397
References 398
20. Generation of biocompatible droplets for in vivo and in vitro measurement of cell-generated mechanical stresses 400
Introduction 401
1. Methods 402
1.1 Generation and Stabilization of Biocompatible Droplets 402
1.1.1 Materials 403
1.1.2 Equipment 403
1.1.3 Method 403
1.2 Functionalization of Droplets 406
1.2.1 Materials 406
1.2.2 Equipment 406
1.2.3 Method 406
1.3 Characterizing the Mechanical Properties of the Droplets 409
1.3.1 Materials 410
1.3.2 Equipment 410
1.3.3 Method 410
1.4 Use of Droplets in Different Applications 413
1.4.1 In vitro 413
1.4.1.1 Single cells and cells in 2D monolayers 413
1.4.1.2 Cell aggregates 413
1.4.2 Ex vivo and in vivo 413
2. Discussion 414
Conclusion 415
References 416
21. Laser induced wounding of the plasma membrane and methods to study the repair process 418
Introduction 419
1. Cell Deposition 420
1.1 Materials 420
1.1.1 Reagents 420
1.1.2 Equipments 420
1.2 Method 421
1.2.1 Cell keeping 421
1.2.2 Cell plating 421
2. Photodamage and Imaging 421
2.1 Materials 421
2.2 Equipments 422
2.3 Method 422
2.3.1 Chamber mounting 422
2.3.2 Cell imaging 423
2.3.3 UV laser calibration and cell damage 424
3. Following Plasma Membrane Damage and Repair 426
3.1 Materials 426
3.2 Method 426
4. Image Analysis 428
4.1 Software 428
4.2 Methods 429
5. Data Analysis 429
5.1 Software 429
5.2 Method 429
6. Discussion 432
6.1 Alternative methods to damage the plasma membrane 432
6.2 Plasma Membrane Repair Alteration 432
6.3 Troubleshooting 433
General Conclusions 433
References 433
22. Electrofusion of giant unilamellar vesicles to cells 436
Introduction 437
1. Preparation of GUVs by Electroformation 438
1.1 Materials 440
1.2 Equipment 440
1.3 Method 440
1.3.1 Preparation of lipid-coated, ITO-coated slides 440
1.3.2 Assembly of the electroformation chamber 440
2. Electrofusion of GUVs to Cells 441
2.1 Materials 442
2.2 Equipment 443
2.3 Method 443
2.3.1 Prepare cell culture 443
2.3.2 Replating cells 443
2.3.3 Electrofusion 444
3. Discussion 445
References 447
23. Measurement and manipulation of cell size parameters in fission yeast 450
Introduction 451
1. Measurement of Size Parameters of Single Fission Yeast Cells 452
1.1 Dynamic Measurement of Cell Size Parameters During Single Spore Growth and Polarization 452
1.1.1 Spore preparation for imaging 452
1.1.2 Imaging 453
1.1.3 Image analysis 453
1.2 Length, Diameter, Surface, and Volume of Dividing Cells 455
1.2.1 Cell preparation for imaging 456
1.2.2 Image analysis 456
2. Microchannel Assay for Cell Diameter Manipulation 457
2.1 Fabricating Microchannels to Manipulate Cell Diameter 457
2.1.1 Photomask design 459
2.1.2 Photolithography 459
2.1.3 Creating PDMS from master 460
2.1.4 Assembling the micro channels 460
2.2 Cell Diameter Manipulation and Imaging 461
Conclusions 462
Acknowledgments 462
References 462
24. Methods for rectifying cell motions in vitro: breaking symmetry using microfabrication and microfluidics 464
Introduction 465
Relevance of Cell Migration In vivo 465
Origin of Symmetry Breaking In vivo and the Need for Controlled In vitro Approaches: Microfabrication and Microfluidics 466
1. Breaking Symmetry with Topography: Fabrication of a Topographical Pattern 466
1.1 Topographical Pattern Design 466
1.1.1 Materials 467
1.1.2 Pattern design 468
1.2 Fabrication of the Micropatterned Substrate 468
1.2.1 Materials 468
1.2.2 Equipment 469
1.2.3 Method 469
2. Breaking Symmetry with Chemical Gradient: Preparation of the Fibronectin Gradient 471
2.1 Microfluidic Chip Design 471
2.1.1 Materials 471
2.1.2 Pattern design 471
2.2 Fabrication of the Microfluidic Chip 471
2.2.1 Materials 471
2.2.2 Equipment 471
2.2.3 Method 472
2.3 Fibronectin Gradient Formation 472
2.3.1 Materials 472
2.3.2 Equipment 473
2.3.3 Method 473
2.3.3.1 Surface activation 473
2.3.3.2 Gradient formation 473
3. Cell Migration Experiments 474
3.1 Materials 476
3.2 Equipment 476
3.3 Method 476
4. Discussion 477
Conclusions 478
References 478
25. Analyzing bacterial movements on surfaces 480
Introduction 481
1. Preparing Bacterial Suspension 482
1.1 Materials 482
1.2 Method 483
2. Tracking Bacteria on Solid Surfaces 484
2.1 Materials 484
2.2 Coating Microscope Coverslips 485
2.3 Mount Coverslip for Microscopy 488
2.4 Imaging and Tracking the Motion of Single Bacteria 489
3. Tracking Bacteria on Cells 491
3.1 Materials 491
3.2 Cleaning and Sterilizing Coverslips 492
3.3 Prepare Coverslips with a Monolayer of Mammalian Cells 492
3.4 Mount Coverslip with Mammalian Cells for Microscopy 492
3.5 Imaging and Tracking the Motion of Single Bacteria 493
4. Discussion Points 494
4.1 Know Your Bacterium 494
4.2 Choose Your Time Frame 494
4.3 Complementary Techniques 494
Conclusions 495
References 495
26. Advances in single-cell experimental design made possible by automated imaging platforms with feedback through segmentation 498
Introduction 499
1. In vitro Experiments where Automation is Important 500
1.1 An Example of Host–Pathogen Interaction Phenotyping: Malaria Parasites and Red Blood Cells 500
1.2 Long-Term Live Imaging in Immune System Cells 501
2. Preparation of Cells Described in this Chapter 502
2.1 Malaria Culture 502
2.2 Macrophages 503
3. Automation Methods 503
3.1 Live Imaging Conditions and Microscope Setup 503
3.2 Development of Tracking Algorithm, Testing on Videos of Multiple Cell Types 503
3.3 Requirements of a Good Image Analysis Solution 504
3.4 A Specific Example of an Effective Segmentation Routine 505
3.5 Connecting Image Analysis to Microscope Hardware 505
3.6 The Automation Concept Deployed on Egress/Invasion, and Tweezers Intervention, in Malaria 508
4. Discussion 510
4.1 Potential Throughput of Single-Cell Tracking Experiments 510
4.2 Implications for Live Imaging 511
5. Outlook 512
Acknowledgments 514
References 514
Volumes in Series 516
Index 528

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Contributors

Sarra Achouri,     Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, United Kingdom

Pedro Almada,     MRC Laboratory for Molecular Cell Biology and Department of Cell and Developmental Biology, University College London, London, UK

Vaishnavi Ananthanarayanan,     Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

Mohammed Ashraf,     Mechanobiology Institute, National University of Singapore, Singapore

Atef Asnacios,     Laboratoire Matières et Systèmes Complexes, Université Paris-Diderot/CNRS, Sorbonne Paris Cité, Paris, France

Martial Balland,     Laboratoire Interdisciplinaire de Physique, UMR 5588, CNRS/Univ. Grenoble-Alpes, Grenoble, France

Matthew E. Berginski,     Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA

Timo Betz,     Centre de Recherche, Institut Curie, Paris Cedex 05, France; Centre National de la Recherche Scientifique, Paris Cedex 05, France; UPMC University Paris VI, Paris, France

Nicolas Biais,     Brooklyn College CUNY, Biology Department, Brooklyn, NY, USA; Graduate Center of CUNY, New York, NY, USA

Maté Biro,     Centenary Institute of Cancer Medicine and Cell Biology, The University of Sydney, Sydney, NSW, Australia

Daria Bonazzi,     Institut Jacques Monod, CNRS UMR, Paris Cedex 13, France

Siobhan A. Braybrook,     The Sainsbury Laboratory, University of Cambridge, Cambridge, UK

G. Wayne Brodland,     Department of Civil and Environmental Engineering, University of Waterloo, Waterloo, ON, Canada

Jan Brugués,     Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany; Max Planck Institute for the Physics of Complex Systems, Dresden, Germany

Nathalie Bufi,     Laboratoire Matières et Systèmes Complexes, Université Paris-Diderot/CNRS, Sorbonne Paris Cité, Paris, France

Matthias Bussonnier,     Centre de Recherche, Institut Curie, Paris Cedex 05, France; Centre National de la Recherche Scientifique, Paris Cedex 05, France; UPMC University Paris VI, Paris, France

Eugenia Cammarota,     Cavendish Laboratory, University of Cambridge, Cambridge, UK

Otger Campàs,     Department of Mechanical Engineering, University of California, Santa Barbara, CA, USA

Kevin J. Chalut,     Cavendish Laboratory, Department of Physics, University of Cambridge, Cambridge, UK; Wellcome Trust/Medical Research Council Stem Cell Institute, Cambridge, UK

Chii J. Chan,     Cavendish Laboratory, Department of Physics, University of Cambridge, Cambridge, UK; Biotechnology Center, Technische Universität Dresden, Tatzberg, Dresden, Germany

Jonathan R. Chubb,     MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London, WC1E 6BT, United Kingdom

Pietro Cicuta,     Cavendish Laboratory, University of Cambridge, Cambridge, UK

Laurent Cognet,     Univ Bordeaux, Laboratoire Photonique Numérique et Nanosciences, Institut d’Optique & CNRS, Talence, France

J. Comelles,     Laboratory of Cell Physics ISIS/IGBMC, CNRS and University of Strasbourg, Strasbourg, France; Development and Stem Cells Program, IGBMC, CNRS, INSERM and University of Strasbourg, Illkirch, France

Vito Conte,     Institute for Bioengineering of Catalonia, Barcelona, Spain

Adam M. Corrigan,     MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London, WC1E 6BT, United Kingdom

Alex J. Crick,     Cavendish Laboratory, University of Cambridge, Cambridge, UK

Franziska Decker,     Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany; Max Planck Institute for the Physics of Complex Systems, Dresden, Germany

Pauline Durand-Smet,     Laboratoire Matières et Systèmes Complexes, Université Paris-Diderot/CNRS, Sorbonne Paris Cité, Paris, France

Andrew E. Ekpenyong,     Biotechnology Center, Technische Universität Dresden, Tatzberg, Dresden, Germany; Department of Physics, Creighton University, Omaha, NE, USA

Kristian Franze,     Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, United Kingdom

Zhenghong Gao,     Univ Bordeaux, Laboratoire Photonique Numérique et Nanosciences, Institut d’Optique & CNRS, Talence, France

Hélène O.B. Gautier,     Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, United Kingdom

Jérémie J. Gautier,     CNRS, Laboratoire d’Enzymologie et Biochimie Structurales, Gif sur Yvette, France

Alexis Gautreau,     CNRS, Laboratoire d’Enzymologie et Biochimie Structurales, Gif sur Yvette, France

Sara Geraldo,     Centre de Recherche, Institut Curie, Paris Cedex 05, France; Centre National de la Recherche Scientifique, Paris Cedex 05, France

Grégory Giannone,     Univ Bordeaux, Interdisciplinary Institute for Neuroscience UMR 5297, CNRS, Bordeaux, France

Jochen Guck,     Biotechnology Center, Technische Universität Dresden, Tatzberg, Dresden, Germany; Cavendish Laboratory, Department of Physics, University of Cambridge, Cambridge, UK

Mukund Gupta,     Mechanobiology Institute, National University of Singapore, Singapore

Ricardo Henriques,     MRC Laboratory for Molecular Cell Biology and Department of Cell and Developmental Biology, University College London, London, UK

Brenton D. Hoffman,     Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA

Kathrin Holtzmann,     Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, United Kingdom

V. Hortigüela,     Biomimetic Systems for Cell Engineering, Institute for Bioengineering of Catalonia (IBEC), Barcelona, Spain; Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina, Zaragoza, Spain

M. Shane Hutson,     Department of Physics and Astronomy, Vanderbilt University, Nashville, TN, USA; Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA; Vanderbilt Institute for Integrative Biosystem Research & Education, Vanderbilt University, Nashville, TN, USA

Donald E. Ingber,     Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA

Ana J. Jimenez,     Institut Curie, Paris Cedex 05, France; CNRS UMR, Paris Cedex 05, France

Kinneret Keren,     Department of Physics, Technion- Israel Institute of Technology, Haifa, Israel; Russell Berrie Nanotechnology Institute, Technion- Israel Institute of Technology, Haifa, Israel; Network Biology Research Laboratories, Technion- Israel Institute of Technology, Haifa, Israel

Leyla Kocgozlu,     Mechanobiology Institute, National University of Singapore, Singapore

Katarzyna S. Kopanska,     Centre de Recherche, Institut Curie, Paris Cedex 05, France; Centre National de la Recherche Scientifique, Paris Cedex 05, France; UPMC University Paris VI, Paris, France

David E. Koser,     Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, United Kingdom

Jurij Kotar,     Cavendish Laboratory, University of Cambridge, Cambridge, UK

Laetitia Kurzawa,     CytoMorpho Lab, Institut de Recherche en Technologie et Science pour le Vivant, LPCV/UMR5168, CEA/INRA/CNRS/Univ. Grenoble-Alpes, Grenoble, France

Andrew S. LaCroix,     Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA

Benoit Ladoux,     Mechanobiology Institute, National University of Singapore, Singapore; Institut Jacques Monod (IJM), CNRS UMR 7592 & Université Paris Diderot, Paris, France

Julie Lafaurie-Janvore,     Institut...

Produkt-Details

• Erscheint lt. Verlag: 29.1.2015
• Sprache: englisch
• Themenwelt: Naturwissenschaften ► Biologie ► Biochemie
• Themenwelt: Naturwissenschaften ► Biologie ► Zellbiologie
• Themenwelt: Technik
• ISBN-10: 0-12-801326-5 / 0128013265
• ISBN-13: 978-0-12-801326-7 / 9780128013267