Biophysical Tools for Biologists (eBook)

Front Cover 1
Methods in Cell Biology 4
Copyright Page 5
Dedication Page 6
Contents 8
Contributors 16
Preface 20
Section I: Fluorescence Methods 22
Chapter 1: In Vivo Applications of Fluorescence Correlation Spectroscopy 24
I. Introduction 25
II. FCS Technology 33
III. Applications of In Vivo FCS 37
IV. Future Directions for In Vivo FCS 46
V. Conclusions 49
Acknowledgments 49
References 49
Chapter 2: Molecular Sensors Based on Fluorescence Resonance Energy Transfer to Visualize Cellular Dynamics 58
I. Introduction 59
II. Basic Principles of FRET-Based Molecular Sensors 59
III. Methods 66
IV. A Case Study of PI3K/Akt Signaling Pathway 73
V. Discussion and Conclusion 75
Acknowledgments 75
References 75
Chapter 3: A Fluorescent Window Into Protein Folding and Aggregation in Cells 80
I. Introduction 81
II. Rationale 82
III. Methods 82
IV. Summary 89
Acknowledgments 90
References 90
Chapter 4: Combining Microfluidics and Quantitative Fluorescence Microscopy to Examine Pancreatic Islet Molecular Physiology 92
I. Introduction 93
II. Rationale 94
III. Methods and Materials 100
IV. Discussion 109
Ackowledgments 110
References 110
Section II: Microscopic Methods 114
Chapter 5: Imaging in Depth: Controversies and Opportunities 116
I. Introduction 117
II. Basic Imaging Methodologies 119
III. Forays Deeper into Depth 121
IV. Discussion: Terms of Resolution 139
V. Summary 141
Acknowledgments 141
References 141
Chapter 6: Principles and Practice in Electron Tomography 150
I. Introduction 151
II. Specimen Preparation 152
III. Data Collection for Electron Tomography 159
IV. Computation of an Electron Tomographic Reconstruction 165
V. Interpretation of Electron Tomographic Reconstructions 172
VI. Summary and Future Directions 182
Acknowledgments 183
References 183
Chapter 7: Total Internal Reflection Fluorescence Microscopy 190
I. Introduction 191
II. Rationale 192
III. Theoretical Principles 196
IV. Combinations of TIRF with Other Techniques 207
V. Optical Configurations and Setup 217
VI. General Experimental Considerations 229
VII. Summary: TIRF Versus Other Optical Section Microscopies 233
Acknowledgments 234
References 234
Chapter 8: Spatiotemporal Dynamics in Bacterial Cells: Real-Time Studies with Single-Event Resolution 244
I. Introduction 245
II. Studying Cellular Dynamics with Single-Event Resolution 246
III. Methods 248
IV. Summary and General Lessons for Following Discrete Events 269
Acknowledgments 270
References 270
Chapter 9: Counting Proteins in Living Cells by Quantitative Fluorescence Microscopy with Internal Standards 274
I. Introduction 275
II. Experimental Methods 278
III. Data Analysis 288
IV. Conclusions 290
Acknowledgments 293
References 293
Chapter 10: Infrared and Raman Microscopy in Cell Biology 296
I. Introduction 297
II. Methods 299
III. Results and Discussion 309
IV. Conclusions 328
Acknowledgment 328
References 328
Chapter 11: Imaging Fluorescent Mice In Vivo Using Confocal Microscopy 330
I. Introduction 331
II. Rationale 331
III. Methods and Materials 342
IV. Discussion and Summary 346
References 347
Chapter 12: Nanoscale Biological Fluorescence Imaging: Breaking the Diffraction Barrier 350
I. Introduction 351
II. Theory and Rationale 356
III. Methods 359
IV. Materials 366
V. Discussion 368
VI. Summary 377
Acknowledgments 377
References 377
Section III: Methods at the In Vitro/In Vivo Interface 380
Chapter 13: Imaging of Cells and Tissues with Mass Spectrometry: Adding Chemical Information to Imaging 382
I. Introduction 383
II. Instrumentation 385
III. Sample Preparation for MSI 393
IV. Image Acquisition and Data Analysis 403
V. Specialized Methods 405
VI. Summary and Future Directions 406
Acknowledgments 407
References 407
Chapter 14: Electron Microscopy of Hydrated Samples 412
I. Introduction 413
II. Basic SEM 413
III. Environmental SEM 417
IV. Wet SEM 422
V. Summary 425
References 427
Section IV: Methods for Diffusion, Viscosity, Force and Displacement 430
Chapter 15: Live-Cell Single-Molecule Force Spectroscopy 432
I. Introduction 433
II. Materials and Instrumentation 441
III. Procedures 442
IV. Pearls and Pitfalls 450
V. Concluding Remarks 451
References 451
Chapter 16: Magnetic Manipulation for Force Measurements in Cell Biology 454
I. Introduction 455
II. Sample Preparation 457
III. Video and Laser-Based Magnetic Systems 460
IV. Calibration of Pole Tips 461
V. Pole Configurations 463
VI. Modes of Magnet Controls 463
VII. Cell Experiments with Magnetics 465
VIII. Driven Bead Rheology of Biologic Fluids 467
IX. Conclusions 470
Acknowledgements 470
References 470
Chapter 17: Application of Laser Tweezers to Studies of Membrane-Cytoskeleton Adhesion 472
I. Introduction 473
II. Materials and Methods 476
III. Tether Force Measurements of the Adhesion Energy Between the Plasma Membrane and the Cortical Cytoskeleton 481
IV. Concluding Remarks 485
References 486
Chapter 18: Sensing Cytoskeletal Mechanics by Ballistic Intracellular Nanorheology (BIN) Coupled with Cell Transfection 488
I. Introduction 489
II. Materials and Instrumentation 494
III. Procedures 498
IV. Pearls and Pitfalls 505
V. Concluding Remarks 506
References 506
Chapter 19: Mechanical Response of Cytoskeletal Networks 508
I. Introduction 509
II. Rheology 510
III. Cross-Linked F-Actin Networks 514
IV. Effects of Microtubules in Composite F-Actin Networks 525
V. Intermediate Filament Networks 533
VI. Conclusions and Outlook 536
Acknowledgments 536
References 537
Chapter 20: Automated Spatial Mapping of Microtubule Catastrophe Rates in Fission Yeast 542
I. Introduction 543
II. Methods 544
III. Results 555
IV. Discussion 556
Acknowledgments 557
References 558
Section V: Techniques for Protein Activity and Protein-Protein Interactions 560
Chapter 21: Quantitative Fluorescence Lifetime Imaging in Cells as a Tool to Design Computational Models of Ran-Regulated Reaction Networks 562
Abbreviations 563
I. Quantitative Imaging and Systems Modeling as a Tool in Cell Biology-The Rationale and Strategy 564
II. Quantitative Detection of Biochemical Interactions by FLIM 567
III. Technical Considerations for FLIM in Live Cells 573
IV. Analysis of the Mitotic RanGTP Gradient Function by FLIM and Computational Modeling 578
V. Materials and Methods 583
References 586
Chapter 22: Quantitation of Protein-Protein Interactions: Confocal FRET Microscopy 590
I. Introduction 591
II. Rationale 592
III. Material and Methods 593
IV. Results and Discussion 603
V. Summary 616
References 616
Section VI: Computational Modeling 620
Chapter 23: Stochastic Modeling Methods in Cell Biology 622
I. Introduction 623
II. Stochastic Methods in Signaling and Genetic Networks 628
III. Molecular Motors and the Inclusion of Biomolecular Structure in Stochastic Models 630
IV. Cytoskeleton and Cytoskeletal Network Structures 634
V. Procedures 638
VI. Discussion and Concluding Remarks 639
VII. Appendix Glossary of Terms 640
References 641
Chapter 24: Computational Modeling of Self-Organized Spindle Formation 644
I. Introduction 645
II. Rationale 646
III. Methods 650
IV. Materials 667
V. Discussion and Summary 668
Acknowledgments 668
VI. Appendix A: Geometry of Dynamic Degrees of Freedom 668
VII. Appendix B: Langevin Calculation Algorithm 669
References 672
Index 674
Volumes in Series 690
Color Plate Section 700