Front Cover	1
	Methods in Cell Biology	4
	Copyright Page	5
	Contents	6
	Contributors	14
	Preface	20
	Part I: Basic Concept and Preparation Culture Substrates for Cell Mechanical Studies	22
	Chapter 1: Basic Rheology for Biologists	24
	I. Introduction and Rationale	25
	II. Rheological Concepts	27
	A. Elasticity	28
	B. Viscosity	29
	C. Oscillatory Measurements	30
	III. Rheological Instrumentation	32
	IV. Experimental Design	34
	A. Stress-Strain Relation	34
	B. Stress Relaxation	35
	C. Creep and Creep Recovery	36
	D. Frequency Sweep	37
	E. Time Sweep	38
	F. Strain or Stress Amplitude Sweeps	39
	G. Rate-Dependent Viscosity	40
	H. Flow Oscillation	41
	V. Sample Preparation	41
	A. Solids	41
	B. Liquids and Gelling Systems	43
	VI. Special Considerations for Biological Samples	43
	A. Biological Polymers	43
	B. Intact Tissue	44
	C. Instrument Selection for Measuring Gelation Kinetics	44
	VII. Conclusions	45
	Glossary	46
	References	47
	Chapter 2: Polyacrylamide Hydrogels for Cell Mechanics: Steps Toward Optimization and Alternative Uses	50
	I. Introduction	51
	II. Principle of the Polyacrylamide Hydrogel	52
	A. Standard Method of Polymerization	52
	B. Light-Induced Initiation of Polymerization	52
	III. Conjugation of Proteins to Polyacrylamide	54
	A. Carbodiimide-Mediated Cross-Linking	55
	B. Activation with Acrylic Acid N-Hydroxysuccinimide Ester	57
	C. Activation with the N-Succinimidyl Ester of Acrylamidohexanoic Acid (N6)	59
	D. Other Protein-Coupling Methods	61
	IV. Optimizing the Placement of Beads for Traction Force Microscopy	61
	V. Manipulation of Gel Geometry	62
	A. Preparation of Polyacrylamide Microbeads	62
	B. Preparation of a Model Three-Dimensional Culture System	63
	VI. Concluding Remarks	65
	References	66
	Chapter 3: Microscopic Methods for Measuring the Elasticity of Gel Substrates for Cell Culture: Microspheres, Microindenters, and Atomic Force Microscopy	68
	I. Introduction	69
	II. Probing with Microspheres Under Gravitational Forces	70
	III. Atomic Force Microscopy	71
	IV. Probing with Spherically Tipped Glass Microindenters	74
	A. Preparation and Calibration of the Spherically Tipped Microindenter	75
	B. Calibration of the Microscope and Micromanipulator	75
	C. Characterization and Calibration of the Microindenter	76
	D. Measurement of the Indentations of Hydrogels in Response to Forces of the Microindenter	78
	E. Data Analysis	82
	F. Discussion	83
	V. Conclusions	85
	References	85
	Chapter 4: Surface Patterning	88
	I. Introduction	89
	II. Patterning with Electrodynamic Instabilities	90
	A. Procedure for Patterning with Electrohydrodynamic Instabilities	92
	B. Cell Migration on Topographic Surfaces	93
	III. Lithography Without a Clean Room	94
	A. Photolithography Basics	95
	IV. Patterning at the Micro- and Nanoscale with Polymer Mixtures and Block Copolymers	101
	A. Principles and Procedures for Controlling Pattern Formation with Polymer Mixtures and Block Copolymers	102
	V. Summary	105
	Acknowledgments	105
	References	106
	Chapter 5: Molecular Engineering of Cellular Environments: Cell Adhesion to Nano-Digital Surfaces	110
	I. Introduction: Sensing Cellular Environments	111
	II. Nano-Digital Chemical Surfaces for Regulating Transmembrane-Receptor Clustering	116
	A. Extended Nanopatterns and Biofunctionalization	116
	B. Micro-Nanopatterns for Spatially Controlled Molecular Clustering	118
	C. Cellular Responses to Nano-Digital and Biofunctionalized Surfaces	122
	D. Local Versus Global Effects of Ligand Density on Cell Adhesion	125
	E. Cell Spreading and Migration on Different Nanopatterns	127
	F. High-Resolution Visualization of Cells in Contact with Biofunctionalized Nanopatterns	128
	III. Outlook for the Future	129
	Acknowledgments	129
	References	130
	Part II: Subcellular Mechanical Properties and Activities	134
	Chapter 6: Probing Cellular Mechanical Responses to Stimuli Using Ballistic Intracellular Nanorheology	136
	I. Introduction	138
	A. Why Cell Mechanics?	138
	B. Particle-Tracking Nanorheology: Measuring the Local Viscoelastic Properties of a Cell by Tracking the Brownian Motion of Individual Nanoparticles Embedded in the Cell	139
	C. Local and Global Viscoelastic Properties of the Cell	140
	D. Interstitial Versus Mesoscale Viscosity of the Cytoplasm	140
	E. Viscoelastic Properties of the Cytoplasm Depend on the Timescale of Applied Forces	141
	F. The Viscoelastic Properties of the Cytoplasm Depend on the Amplitude of Applied Forces	142
	G. Measurements at the Cell Surface Versus in the Cytoplasm	142
	H. Methods of Delivery of Nanoparticles to the Cytoplasm	144
	I. BIN: Proof of Principle	145
	J. Intracellular Micromechanics of Cells in 3D Matrix	146
	II. Materials and Instrumentation	148
	A. Preparation of Nanoparticles	148
	B. Cell Culture	148
	C. Ballistic Injection of Nanoparticles	150
	D. Encapsulation of Cells in a 3D Matrix	150
	E. Imaging of Fluctuating Nanoparticles Embedded in the Cell	150
	III. Procedures	150
	A. Preparation of Nanoparticles	150
	B. Ballistic Injection of Nanoparticles	151
	C. Cell Seeding and Encapsulation in a 3D Matrix	152
	D. BIN Analysis	153
	IV. Pearls and Pitfalls	156
	V. Concluding Remarks	157
	A. Unique Advantages of BIN	157
	B. Advantages of Traditional Particle-Tracking Nanorheology Are Maintained by BIN	157
	Acknowledgments	158
	References	158
	Chapter 7: Multiple-Particle Tracking and Two-Point Microrheology in Cells	162
	I. Introduction	163
	II. Principles of Passive Tracer Microrheology	167
	A. Conventional Passive Microrheology	167
	B. Expected Tracer Motion and Tracking Performance	168
	C. Two-Point Microrheology	169
	III. Multiple-Particle Tracking Algorithms	170
	A. Image Restoration	171
	B. Locating Possible Particle Positions	171
	C. Eliminating Spurious or Unwanted Particle Trajectories	172
	D. Linking Positions into Trajectories	173
	E. Available Software Packages and Computing Resources	175
	IV. Computing Rheology from Tracer Trajectories	176
	A. Computing Mean-Squared Displacements	176
	B. Computing Two-Point Mean-Squared Displacements	178
	C. Applying Automated Image Analysis for Statistics	179
	D. Converting MSDs to Rheology	180
	V. Error Sources in Multiple-Particle Tracking	182
	A. Random Error (Camera Noise)	183
	B. Systematic Errors	184
	C. Dynamic Error	186
	D. Sample Drift, Computational Detrending, and Its Limitations	187
	E. Effects of Measurement Errors on the MSD	188
	VI. Instrument Requirements for High-Performance Tracking	189
	A. Isolation from Vibration, Acoustic Noise, and Thermal Drift	189
	B. Microscopy-Generation of High-Contrast Tracer Images	189
	C. Using Low-Noise, Non-interlaced Camera	191
	D. Using High-Intensity, Filtered Illuminator	192
	E. Using Synthetic Tracers to Increase Visibility	193
	VII. Example: Cultured Epithelial Cells	193
	A. Particle-Tracking Results for TC7 Epithelial Cells	193
	B. TPM of TC7 Epithelial Cells	195
	C. Computing Stress Fluctuation Spectra	197
	VIII. Conclusions and Future Directions	198
	References	198
	Chapter 8: Imaging Stress Propagation in the Cytoplasm of a Living Cell	200
	I. Introduction	201
	II. Detecting External Stress-Induced Displacements in the Cytoplasm	202
	A. Green Fluorescent Protein Transfection and Cell Culture	202
	B. Application of Periodic Mechanical Stress	203
	C. Image Acquisition	203
	D. Image Partitioning	204
	E. Array Shifting	205
	F. Image Match Searching	206
	G. Synchronous Displacement	206
	H. Signal-to-Noise Ratio	208
	III. Imaging Displacement and Stress Maps in a Live Cell	209
	A. Quantifying Displacement Maps	209
	B. Computing Stress Maps from Displacement Maps	211
	C. Modulation of Stress Distribution Within a Living Cell	211
	D. Application of Mechanical Loads in Any Direction Using 3D-MTC	213
	E. Mechanical Anisotropic Signaling to the Cytoskeleton and to the Nucleolus	216
	F. Limitations of 3D-MTC	218
	IV. Future Prospects	218
	Acknowledgments	218
	References	219
	Chapter 9: Probing Intracellular Force Distributions by High-Resolution Live Cell Imaging and Inverse Dynamics	220
	I. Introduction	221
	II. Methods	223
	A. Actin Cytoskeleton Mechanics in Cell Protrusion	223
	B. Force Reconstruction	233
	C. Probing Heterogeneous Network Elasticity with Speckle Microscopy	242
	III. Summary	248
	IV. Appendix	249
	A. Solution of the Inverse Problem	249
	Acknowledgments	252
	References	252
	Chapter 10: Analysis of Microtubule Curvature	258
	I. Introduction	259
	II. Rationale	261
	III. Raw Data Collection	263
	A. Point-Click Method	264
	B. Semiautomated Methods	264
	C. Data Collection Errors	265
	IV. Validation Strategy	266
	A. Modeling of Semiflexible Polymers	268
	B. Generation of Simulated Data	269
	C. Validation of Semiflexible Polymer Simulation	274
	V. Curvature Estimation Methods	277
	A. Three-Point Method	277
	B. Shape-Fitting Method	277
	C. Constructing the Curvature Distribution	278
	VI. Results	279
	A. Three-Point Method	279
	B. Shape-Fitting Method	283
	VII. Discussion	285
	VIII. Conclusions	286
	Acknowledgments	286
	References	287
	Chapter 11: Nuclear Mechanics and Methods	290
	I. Introduction	291
	II. Experimental Methods for Probing Nuclear Mechanical Properties	294
	A. Isolation of Individual Nuclei	294
	B. Micropipette Aspiration Experiments	296
	C. Substrate Strain Experiments	298
	D. Compression Experiments	301
	E. Indentation by AFM	306
	F. Particle-Tracking Microrheology	307
	G. Microneedle-Imposed Extension	308
	III. Discussion and Prospects	309
	IV. Outlook	311
	References	312
	Part III: Cellular and Embryonic Mechanical Properties and Activities	316
	Chapter 12: The Use of Gelatin Substrates for Traction Force Microscopy in Rapidly Moving Cells	318
	I. Introduction	319
	II. Rationale	320
	III. Methods	321
	A. Preparation of Gelatin Substrates	321
	B. Plating Cells onto a Gelatin Substrate	323
	C. Data Collection and Analysis	324
	D. Trouble-Shooting Guide	326
	IV. Applications of the Gelatin Traction Force Assay to Study Mechano-signal Transduction in Moving Keratocytes	328
	V. Other Applications and Future Directions	330
	VI. Summary	331
	Acknowledgments	331
	References	331
	Chapter 13: Microfabricated Silicone Elastomeric Post Arrays for Measuring Traction Forces of Adherent Cells	334
	I. Introduction	335
	II. Microfabrication of the Micropost Arrays	337
	A. Standard Photolithography	338
	B. SU-8 Photolithography for Micropost Arrays	339
	C. Soft Lithography	341
	D. Troubleshooting anf Helpful Suggestions	342
	III. Characterization of Micropost Spring Constant	342
	A. Beam-Bending Theory	342
	B. Measurement of Micropost Stiffness	343
	IV. Analysis of Traction Forces Through Micropost Deflections	344
	A. Substrate Preparation	344
	B. Staining and Microscopy of Micropost Arrays	346
	C. Image Analysis Techniques	346
	V. Experimental Applications of Microposts and Discussion	347
	Acknowledgments	348
	References	348
	Chapter 14: Cell Adhesion Strengthening: Measurement and Analysis	350
	I. Introduction	351
	II. The Cell Adhesion Process	351
	III. Measurement Systems for Adhesion Characterization	352
	IV. Hydrodynamic Assay for Quantifying Adhesion Strength	355
	A. Experimental Design	355
	B. Interpretation of Adhesion Strength Results	358
	V. Quantitative Biochemical Methods for Adhesion Analysis	359
	A. Quantification of Bound Integrin	359
	B. Wet-Cleaving. Assay for Localized FA Protein Quantification	359
	C. Immunofluorescence Staining and Quantification	360
	VI. Simple Mathematical Modeling of Adhesion Strengthening Mechanics	362
	A. Resolving Forces for a Cell Under Hydrodynamic Shear	362
	B. Mathematical Analysis of Adhesion Strengthening Mechanics	364
	VII. Discussion	365
	Acknowledgments	365
	References	365
	Chapter 15: Studying the Mechanics of Cellular Processes by Atomic Force Microscopy	368
	I. Introduction	369
	II. Instrumentation and Operation Modes	370
	A. Principal Components	370
	B. Cantilevers	371
	C. Combination of Optical Microscopy and AFM	373
	III. Operating Modes	374
	A. Imaging Modes	374
	B. Force Curve Mode	378
	C. Force Volume Mode	379
	IV. Investigations of Live Cells	379
	A. Imaging	379
	B. Measuring Stiffness	379
	C. Dynamics of Cellular Mechanical Properties	386
	D. Effects of Cell Stiffness on AFM Imaging	388
	V. Outlook	389
	Acknowledgments	390
	References	390
	Chapter 16: Using Force to Probe Single-Molecule Receptor-Cytoskeletal Anchoring Beneath the Surface of a Living Cell	394
	I. Generic Methods and Physical Foundations	395
	A. Using Force to Probe Single-Molecule Interactions	395
	B. Testing Bonds on Solid Substrates	396
	C. Simple Physics of Breaking a Bond	398
	D. Forcing Bonds to Dissociate Faster Than Their Spontaneous Off Rate	400
	E. Force Ramp Method and Examples of Testing Protein-Protein Interactions In Vitro	401
	II. Probing Bonds at Cell Surfaces	404
	A. Phenomenology of Unbinding Events	404
	B. Experimental Frustrations	407
	C. Most Likely Site of Molecular Unbinding When Probing Bonds at Cell Surfaces	408
	D. Adhesive Failure Without Membrane-Cytoskeletal Separation	409
	E. Membrane-Cytoskeletal Unbinding Followed by Adhesive Detachment	411
	F. Overlapping Bond Failure Processes	412
	III. Future Challenge and Opportunity	414
	Acknowledgments	416
	References	416
	Chapter 17: High-Throughput Rheological Measurements with an Optical Stretcher	418
	I. Introduction	419
	II. Rationale	422
	III. Methods	423
	A. Basic Experimental Setup	423
	B. Preparation of Cells	425
	C. Measurement Process	425
	D. Computer Control of the Measurement	426
	E. Image Analysis	427
	F. Interpretation of the Data	428
	IV. Additional Notes on Equipment	433
	A. Optical Fibers	433
	B. Laser Source	434
	C. Microfluidic Chip	435
	D. Microscopy Technique	439
	E. Camera	440
	V. Discussion	440
	VI. Summary	442
	Acknowledgments	442
	References	442
	Chapter 18: Measuring Mechanical Properties of Embryos and Embryonic Tissues	446
	I. Introduction	447
	A. Biomechanics and Developmental Biology	447
	B. Why Do We Want to Understand the Mechanical Basis of Morphogenesis?	448
	II. Applying and Measuring Forces of 10 nN to 10 muN	449
	III. Nanonewton Force Apparatus: Parts, Function, and Operation	452
	IV. Preparation of Tissue Samples	453
	V. Measurement of the Time-Dependent Elasticity of Embryos or Tissue Explants	455
	VI. Spring and Dashpot Models of Viscoelasticity Represent More Complex Structural Sources	456
	VII. Challenges of Working with Embryonic Tissues	456
	VIII. Use of Standard Engineering Terms and Units	458
	IX. Future Prospects	458
	References	458
	Part IV: Mechanical Stimuli to Cells	462
	Chapter 19: Tools to Study Cell Mechanics and Mechanotransduction	464
	I. Introduction	465
	II. Control of Cell Shape, Cytoskeletal Organization, and Cell Fate Switching	467
	A. Microcontact Printing of Micropatterned Substrates for Cell Culture	468
	B. Application Notes on Microcontact Printing	470
	C. Extension and Future Development of Microcontact Printing	474
	III. Probing Cell Mechanics, Cytoskeletal Structure, and Mechanotransduction	475
	A. Magnetic Twisting Cytometry (MTC)	476
	B. Applications of MTC	479
	C. Extension and Future Development of MTC	480
	D. Magnetic Pulling Cytometry (MPC)	481
	E. Applications of MPC	488
	IV. Discussion and Future Implications	488
	References	490
	Chapter 20: Magnetic Tweezers in Cell Biology	494
	I. Introduction	495
	II. Physics of Magnetic Tweezers	496
	III. Magnetic Field Considerations	498
	A. Sources of Magnetic Field	498
	B. Magnet Pole Design	499
	IV. Magnetic Particle Selection	500
	V. Basic Solenoid Apparatus	502
	VI. Force Calibration	503
	A. Calibration Sample Protocol	505
	B. Calibration Procedure	506
	C. Direction of Magnetic Force	506
	D. Bread-Tracking System	507
	E. Data Interpretation	507
	VII. Experimental Procedures	508
	References	512
	Chapter 21: Optical Neuronal Guidance	516
	I. Introduction	517
	A. Neuron Structure	517
	B. Neuronal Cells in Development	517
	C. Growth Cone Movement and Guidance	519
	D. Existing Guidance Methods	521
	II. Apparatus	522
	A. Laser Light Sources	522
	B. Laser Light Control Elements	523
	C. Microscope Irradiation and Imaging	525
	D. Cell Culture System	525
	E. Adapting Existing Systems for Optical Guidance	529
	III. Experiments	530
	A. Turns	530
	B. Accelerated Growth	531
	C. Bifurcations	532
	D. Other Observations	532
	IV. Plausible Mechanisms of Optical Guidance	534
	A. Filopodial Asymmetries	534
	B. Retrograde Flow	536
	C. Actin Polymerization via Membrane Tweezing	536
	D. Laser-Induced Heating	536
	V. Summary	537
	Acknowledgments	538
	References	538
	Chapter 22: Microtissue Elasticity: Measurements by Atomic Force Microscopy and Its Influence on Cell Differentiation	542
	I. Introduction	543
	II. AFM in Microelasticity Measurements	547
	A. AFM Probing and Analysis	547
	B. General Issues in Sample Preparation	551
	III. Materials Characterization	552
	A. Artificial Matrices	552
	B. Cell-Secreted Matrices	557
	C. Passive Tissue Elasticity	559
	IV. Assessing Mechanical Influences on Cells	562
	References	563
	Chapter 23: Demystifying the Effects of a Three-Dimensional Microenvironment in Tissue Morphogenesis	568
	I. Introduction	569
	II. Rationale	571
	A. Stromal-Epithelial Interactions	571
	B. ECM Mechanics and Epithelial Behavior	572
	C. 3D Organotypic Model Systems	577
	III. Methods	579
	A. Engineered Cell/Tissue Explants	579
	B. Isolation of Bulk Proteins	586
	C. Isolation of Bulk mRNA	588
	D. Rapid Protein Isolation Techniques	590
	E. Immunofluorescence	592
	IV. Materials	594
	A. Engineering Tissue Explants	594
	B. Isolation of Bulk Proteins	596
	C. Isolation of Bulk mRNA	596
	D. Rapid Protein Isolation Techniques	597
	E. Immunofluorescence	597
	V. Discussion	598
	Acknowledgments	600
	References	601
	Index	606
	Volumes in Series	622