Paluch Biophysical Methods in Cell Biology

1. Auflage 2015
ISBN: 978-0-12-801326-7
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, Band Volume 125, 568 Seiten

Reihe: Methods in Cell Biology

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

Autoren/Hrsg.

Paluch, Ewa

Inhaltsverzeichnis

1;Front Cover;1
2;Methods in Cell Biology;2
3;Series Editors;3
4;Methods in Cell Biology
;4
5;Copyright
;5
6;Contents;6
7;Contributors;16
8;Preface;26
9;1. Single-molecule imaging of cytoplasmic dynein in vivo;28
9.1;Introduction;29
9.2;1. Visualization of Cytoplasmic Dynein In Vivo;29
9.2.1;1.1 Background;29
9.2.2;1.2 Experiment;30
9.2.2.1;1.2.1 Preparation of fission yeast zygotes;30
9.2.2.2;1.2.2 Observation of dyneins in the cytoplasm;31
9.2.2.3;1.2.3 Observation of dyneins on the microtubule;32
9.2.2.4;1.2.4 Discussion;32
9.2.2.4.1;1.2.4.1 Prebleaching to observe dyneins with higher SNR;32
9.2.2.4.2;1.2.4.1 Prebleaching to observe dyneins with higher SNR;32
9.2.2.4.3;1.2.4.2 Estimation of penetration depth of HILO;33
9.2.2.4.4;1.2.4.2 Estimation of penetration depth of HILO;33
9.3;2. Image Analysis;34
9.3.1;2.1 Tracking of Single Molecules;34
9.3.2;2.2 Confirmation of Single-Molecule Imaging;35
9.3.3;2.3 Analysis of Dynein Movement;36
9.4;3. Conclusion;37
9.5;4. Methods;37
9.5.1;4.1 Cell Culture;37
9.5.2;4.2 Preparation of Samples for Imaging;37
9.5.3;4.3 Microscopy;37
9.6;References;38
10;2. Single-molecule imaging in live cell using gold nanoparticles;40
10.1;Introduction and Rationale;41
10.2;1. Gold Nanoparticle Synthesis and Functionalization;42
10.2.1;1.1 Materials;42
10.2.1.1;1.1.1 List of chemicals required for the nanoparticle synthesis and functionalization;42
10.2.2;1.2 Nanoparticle Synthesis;43
10.2.3;1.3 Nanoparticle Functionalization with Nanobodies;45
10.2.4;1.4 Sample Characterization;46
10.2.4.1;1.4.1 Absorption spectra;46
10.2.4.2;1.4.2 Transmission electron microscope;46
10.2.4.3;1.4.3 Agarose gel electrophoresis;46
10.3;2. Photothermal Imaging;47
10.3.1;2.1 Materials;47
10.3.2;2.2 Principle;47
10.3.3;2.3 Experimental Setup;48
10.3.4;2.4 Resolution and Sensitivity;49
10.3.4.1;2.4.1 Resolution;49
10.3.4.2;2.4.2 Sensitivity;49
10.4;3. Live Cell Imaging;49
10.4.1;3.1 Cell surface labeling;49
10.4.2;3.2 2D Single-Particle Tracking;51
10.4.3;3.3 Particle Internalization;52
10.5;Conclusion;53
10.6;Acknowledgments;53
10.7;References;53
11;3. Quantitative measurement of transcription dynamics in living cells;56
11.1;1. Visualizing Transcription in Living Cells;57
11.2;2. Experimental Protocols;59
11.3;3. Cell Segmentation;59
11.4;4. Measuring Spot Intensity;62
11.5;5. Correcting for Background MS2-GFP Level;62
11.6;6. Tracking Algorithms;64
11.7;7. Additional Cell Properties;66
11.8;8. Summary;66
11.9;References;67
12;4. An easy-to-use single-molecule speckle microscopy enabling nanometer-scale flow and wide-range lifetime measurement of cell ...;70
12.1;Introduction;71
12.2;1. Methods;73
12.2.1;1.1 Preparation of Actin Probes;73
12.2.1.1;1.1.1 Required materials;74
12.2.1.2;1.1.2 Procedure for DyLight NHS ester labeling of actin;74
12.2.2;1.2 Electroporation Method for Delivery of DL-Actin to XTC Cells;75
12.2.2.1;1.2.1 Required materials;76
12.2.2.2;1.2.2 Procedure for electroporation of DL-actin into XTC cells;77
12.2.3;1.3 SiMS Imaging;78
12.2.3.1;1.3.1 Required materials;78
12.2.3.2;1.3.2 Examples of microscopy setups;79
12.2.3.3;1.3.3 Procedure for the SiMS imaging of DL-actin loaded XTC cells;79
12.2.4;1.4 Data Analysis;80
12.2.4.1;1.4.1 Nanometer-scale displacement measurement;81
12.2.4.2;1.4.2 Simultaneous analysis of actin dynamics with diverse timescales;81
12.3;2. Perspectives;83
12.4;Acknowledgments;84
12.5;References;84
13;5. Dissecting microtubule structures by laser ablation;88
13.1;Introduction;89
13.2;1. Theoretical Framework;90
13.3;2. Microtubule Organization Measurements;95
13.3.1;2.1 Extract and Sample Preparation;95
13.3.2;2.2 Microscopy and Laser Ablation Setup;95
13.3.3;2.3 Data Acquisition and Analysis;95
13.4;Discussion and Conclusion;99
13.5;Acknowledgments;100
13.6;References;100
14;6. Quantifying mitochondrial content in living cells;104
14.1;Introduction;105
14.2;1. Basic Protocol (96-Well Glass Bottom Plate);106
14.2.1;1.1 Sample Preparation;106
14.2.1.1;1.1.1 Materials;106
14.2.1.2;1.1.2 Yeast culturing;107
14.2.1.3;1.1.3 96-well glass plate preparation and cell plating;108
14.2.2;1.2 Image Acquisition;108
14.2.2.1;1.2.1 3D spinning-disk confocal microscope setup;108
14.2.2.2;1.2.2 Imaging;108
14.2.3;1.3 Image Processing;109
14.2.3.1;1.3.1 Data preparation;109
14.2.3.2;1.3.2 Running data through MitoGraph software;110
14.2.3.3;1.3.3 Interpreting the data;111
14.2.3.3.1;1.3.3.1 Group files (one per folder);111
14.2.3.3.2;1.3.3.2 Individual files (one for each TIFF file in the folder);112
14.3;2. Alternate Protocol (CellASIC Microfluidic Flow Chamber);113
14.3.1;2.1 Materials;113
14.3.2;2.2 Yeast Culturing Step;113
14.3.3;2.3 Cell Loading;113
14.4;3. Important Considerations for Successful MitoGraph Performance;114
14.4.1;3.1 Validation: Reproducibility and Accuracy;114
14.4.2;3.2 Optimal Magnification;114
14.4.3;3.3 Signal versus Background Noise Intensity Requirements;115
14.4.4;3.4 Effects of Spherical Aberration;115
14.4.5;3.5 Pros and Cons of “Surface Volume” and “Skeleton Volume”;116
14.5;4. Beyond Wild-type Mitochondria in Budding Yeast Imaged with Spinning-Disk Confocal Microscopy;116
14.5.1;4.1 Other Microscopy Modalities;116
14.5.1.1;4.1.1 Epifluorescence microscope;116
14.5.1.2;4.1.2 Laser-scanning confocal microscope;117
14.5.2;4.2 Mitochondrial Morphology Mutant Cells;117
14.5.3;4.3 Mitochondrial Networks in Nonyeast Cells;119
14.6;Acknowledgments;120
14.7;Supplementary data;120
14.8;References;120
15;7. High-content 3D multicolor super-resolution localization microscopy;122
15.1;Introduction;123
15.2;The Basis of an SMLM-Imaging Experiment;126
15.3;Hybridizing SMLM with High-Content Imaging;128
15.4;1. Sample Preparation;129
15.4.1;1.1 Equipment;129
15.4.2;1.2 Materials;129
15.4.3;1.3 Method;130
15.4.3.1;1.3.1 Cleaning slides and coverslips;130
15.4.3.2;1.3.2 Labeling with primary antibodies;131
15.4.3.3;1.3.3 Seeding the cells;132
15.4.3.4;1.3.4 Immunofluorescence;132
15.4.3.5;1.3.5 Coverslip mounting;133
15.5;2. Imaging Acquisition and Image Analysis;134
15.5.1;2.1 Equipment;134
15.5.2;2.2 Software;135
15.5.3;2.3 Method;135
15.5.3.1;2.3.1 Acquisition;135
15.5.3.2;2.3.2 Single-particle detection and reconstruction;137
15.5.4;2.4 Important considerations;137
15.5.4.1;2.4.1 Detectors used for the acquisition;137
15.5.4.2;2.4.2 Single-molecule detection and localization;138
15.5.4.3;2.4.3 SR drift correction and chromatic realignment;139
15.5.4.4;2.4.4 SR estimation and image reconstruction;139
15.6;Conclusions and Outlook;139
15.7;Acknowledgments;140
15.8;References;140
16;8. Superresolution measurements in vivo: Imaging Drosophila embryo by photoactivated localization microscopy;146
16.1;Introduction;147
16.2;1. Embryo Preparation;148
16.2.1;1.1 Materials;148
16.2.2;1.2 Embryo Staging and Dechorionation;149
16.2.3;1.3 Fixation;149
16.2.4;1.4 Devitellinization;151
16.3;2. Sample Mounting;151
16.3.1;2.1 Materials;151
16.3.2;2.2 Sample Mounting for High-Resolution Imaging;151
16.3.2.1;2.2.1 Coverslip preparation;151
16.3.2.2;2.2.2 Sample mounting;152
16.3.3;2.3 Single-Molecule Sample Preparation;152
16.4;3. Optical Setup;152
16.4.1;3.1 Principles of PALM;152
16.4.1.1;3.1.1 Photoactivable fluorescent protein;152
16.4.1.2;3.1.2 Principles of PALM;153
16.4.2;3.2 Basic Optical Setup;153
16.4.2.1;3.2.1 Illumination;153
16.4.2.2;3.2.2 Optical components;155
16.4.2.3;3.2.3 Optical schemes;155
16.4.2.4;3.2.4 3-D detection;155
16.4.3;3.3 Custom Optimization;157
16.4.3.1;3.3.1 Optimization the illumination;157
16.4.3.2;3.3.2 Synchronization;157
16.4.3.3;3.3.3 Stabilization;159
16.5;4. Imaging;159
16.6;5. Data Analysis;160
16.6.1;5.1 Single-Molecule Detection;160
16.6.2;5.2 Single-Molecule Localization;161
16.6.3;5.3 Drift Characterization and Correction;163
16.6.4;5.4 Estimation of PALM Localization Precision;163
16.6.5;5.5 PALM Data Visualization;165
16.6.6;5.6 Single-Molecule Counting;166
16.6.6.1;5.6.1 Characterization and correction for photoblinking;166
16.6.6.2;5.6.2 Estimation of labeling fraction;166
16.7;6. Summary and Outlook;167
16.8;Acknowledgments;167
16.9;References;167
17;9. Refractive index measurements of single, spherical cells using digital holographic microscopy;170
17.1;Introduction;171
17.2;1. Setup;173
17.3;2. Measurement Preparation;175
17.3.1;2.1 Materials;175
17.3.2;2.2 Method;175
17.3.3;2.3 Comments;176
17.4;3. Data Analysis;176
17.4.1;3.1 Determination of the Phase Distribution;177
17.4.2;3.2 Determination of the RI;179
17.5;4. Discussion;181
17.5.1;4.1 Setup Alterations;181
17.5.2;4.2 Assumption of Spherical Shape for Refractive Index Determination;181
17.5.3;4.3 Analysis Alterations;182
17.6;5. Summary;183
17.7;References;184
18;10. Construction, imaging, and analysis of FRET-based tension sensors in living cells;188
18.1;Introduction;189
18.2;1. Design, Production, and Validation of Tension Sensors;190
18.2.1;1.1 Required Constructs;190
18.2.2;1.2 Materials for Tension Sensor Creation;192
18.2.3;1.3 Design and Production of Constructs;193
18.2.3.1;1.3.1 Restriction enzyme-based cloning;193
18.2.3.1.1;1.3.1.1 Methods;193
18.2.3.2;1.3.2 Megaprimer-based overlap extension;195
18.2.3.2.1;1.3.2.1 Methods;195
18.2.3.3;1.3.3 Gibson assembly;196
18.2.3.3.1;1.3.3.1 Methods;197
18.2.4;1.4 Biochemically Validating a Tension Sensor;197
18.3;2. Imaging of FRET-Based Biosensors;198
18.3.1;2.1 Key Concepts of FRET Imaging;198
18.3.2;2.2 Materials and Equipment;199
18.3.2.1;2.2.1 Reagents;199
18.3.2.2;2.2.2 Equipment;200
18.3.3;2.3 Preparation for and Imaging of FRET-Based Tension Sensors;200
18.3.3.1;2.3.1 Experimental sample preparation;200
18.3.3.1.1;2.3.1.1 Methods;201
18.3.3.2;2.3.2 Detection of common imaging artifacts;201
18.3.3.2.1;2.3.2.1 Methods;201
18.3.3.3;2.3.3 Imaging of tension sensors and control constructs;202
18.3.3.3.1;2.3.3.1 Methods;203
18.4;3. Methods of Analysis of FRET Images;203
18.4.1;3.1 Quantification and Correction of Common Imaging Artifacts;203
18.4.1.1;3.1.1 Methods;205
18.4.1.2;3.1.2 Results;208
18.4.2;3.2 Examples of Common Errors in FRET Imaging;209
18.5;4. Summary;210
18.6;References;211
19;11. Single-cell mechanics: The parallel plates technique;214
19.1;Introduction;215
19.2;1. Experimental Setup;216
19.3;2. Microplates;218
19.3.1;2.1 Microplate Fabrication;219
19.3.1.1;2.1.1 Materials;219
19.3.1.2;2.1.2 Equipment;219
19.3.1.3;2.1.3 Method;219
19.3.2;2.2 Microplate Calibration;220
19.3.2.1;2.2.1 Materials;221
19.3.2.2;2.2.2 Method;221
19.3.3;2.3 Microplate Cleaning;221
19.3.3.1;2.3.1 Materials;221
19.3.3.2;2.3.2 Equipment;222
19.3.3.3;2.3.3 Method;222
19.3.4;2.4 Microplate Coating;223
19.3.4.1;2.4.1 Materials;223
19.3.4.2;2.4.2 Equipment;223
19.3.4.3;2.4.3 Method;223
19.3.4.4;2.4.4 Possible modifications for different coatings;223
19.4;3. Cell Preparation;224
19.4.1;3.1 Materials;224
19.4.2;3.2 Equipment;224
19.4.3;3.3 Method;224
19.5;4. Experimental Protocols;225
19.5.1;4.1 Cell Capture;225
19.5.2;4.2 Calibration of the Optical Sensor;226
19.5.3;4.3 Single-Cell Traction Force Measurements;226
19.5.3.1;4.3.1 Simple traction;226
19.5.3.2;4.3.2 Real-time single-cell response to stiffness;227
19.5.4;4.4 Single-Cell Rheology;227
19.5.4.1;4.4.1 Dynamic mechanical analysis;227
19.5.4.2;4.4.2 Creep experiment;232
19.5.4.3;4.4.3 Relaxation experiment;232
19.6;5. Discussion;233
19.7;Supplementary Data;235
19.8;References;236
20;12. Atomic force microscopy-based force measurements on animal cells and tissues;238
20.1;Introduction;239
20.2;1. Experimental Setup;240
20.2.1;1.1 Setup Design;240
20.2.2;1.2 Cantilever Calibration;241
20.3;2. Sample Preparation;243
20.3.1;2.1 Animal Pretreatment;243
20.3.2;2.2 Preparation of Measurement Buffers;244
20.3.3;2.3 Sample Immobilization;244
20.4;3. AFM and Optical Imaging;245
20.5;4. Measuring Cell and Tissue Stiffness;247
20.5.1;4.1 Important Parameters for Indentation Measurements;249
20.5.2;4.2 Analysis of Indentation Experiments;250
20.6;5. Measuring Adhesion;252
20.6.1;5.1 Chemical Force Microscopy;253
20.6.2;5.2 Single-Molecule Force Spectroscopy;254
20.6.3;5.3 Single-Cell Force Spectroscopy;255
20.7;6. Further Applications;255
20.8;Conclusions;257
20.9;Acknowledgments;258
20.10;References;258
21;13. Measuring the elasticity of plant cells with atomic force microscopy;264
21.1;Introduction;265
21.2;1. Sample preparation and mounting;265
21.2.1;1.1 Materials;266
21.2.2;1.2 Method;266
21.3;2. Atomic force microscopy;267
21.3.1;2.1 Material;267
21.3.2;2.2 Method;267
21.3.2.1;2.2.1 Generalized method;267
21.4;3. Experimental design;270
21.5;4. Discussion;277
21.6;5. Notes;277
21.7;Acknowledgments;279
21.8;References;280
22;14. Dual pipette aspiration: A unique tool for studying intercellular adhesion;282
22.1;Introduction;283
22.2;1. Overview of the DPA Setup;284
22.3;2. Preparing the Pipettes;284
22.3.1;2.1 Materials;284
22.3.2;2.2 Equipment;284
22.3.3;2.3 Methods;286
22.4;3. Preparing the Aspiration Assay;286
22.4.1;3.1 Materials;287
22.4.2;3.2 Equipment;287
22.4.3;3.3 Methods;287
22.5;4. Cell Micromanipulation;288
22.5.1;4.1 Materials;288
22.5.2;4.2 Equipment;288
22.5.3;4.3 Methods;288
22.6;5. Discussion;291
22.7;General Conclusions;292
22.8;References;293
23;15. Measurement of cell traction forces with ImageJ;296
23.1;Introduction;297
23.2;1. Force Measurement Principle;298
23.3;2. Critical Experimental Parameters;301
23.4;3. Critical Numerical Parameters;303
23.5;4. Preparation of Patterned Polyacrylamide Gels with Fiducial Markers;307
23.5.1;4.1 Materials;307
23.5.2;4.2 Equipment;307
23.5.3;4.3 Coating of 24×24mm Glass Coverslips with ECM;307
23.5.4;4.4 Silanization of 20×20mm Glass Coverslips;309
23.5.5;4.5 Polymerization of Polyacrylamide;309
23.6;5. Image Acquisition;310
23.7;6. Image Analysis, Estimation of Displacement, and Traction Force Fields;310
23.7.1;6.1 Preparation of ImageJ Software;310
23.7.2;6.2 Generation of the Parameter File;310
23.7.3;6.3 Measurement of the Cell Traction Energy;311
23.7.3.1;6.3.1 Displacement and force vectors;312
23.7.3.2;6.3.2 Mechanical energy stored in gel deformation;312
23.8;Conclusion;312
23.9;Supplementary Data;313
23.10;References;313
24;16. Micropillar substrates: A tool for studying cell mechanobiology;316
24.1;Introduction;317
24.2;1. Substrate Fabrication;319
24.2.1;1.1 Materials;320
24.2.2;1.2 Equipment;321
24.2.3;1.3 Methods;321
24.2.3.1;1.3.1 Fabrication of silicon wafer;321
24.2.3.2;1.3.2 Silanization of silicon wafer;322
24.2.3.3;1.3.3 Preparation of micropillar substrates;323
24.3;2. Substrate Characterization;323
24.4;3. Substrate Functionalization;325
24.4.1;3.1 Materials;325
24.4.2;3.2 Equipment;327
24.4.3;3.3 Methods;327
24.4.3.1;3.3.1 Preparation of Cy5.5 conjugated fibronectin;327
24.4.3.2;3.3.2 Microcontact printing of fibronectin;327
24.4.3.3;3.3.3 Adapted method for high-aspect ratio micropillars;328
24.5;4. Cells Seeding and Imaging;328
24.5.1;4.1 Materials;329
24.5.2;4.2 Equipment;329
24.5.3;4.3 Methods;329
24.6;5. Image Analysis and Evaluation of Traction Force;330
24.7;6. Discussion and Perspectives;332
24.8;Acknowledgments;333
24.9;References;333
25;17. Mapping forces and kinematics during collective cell migration;336
25.1;Introduction;337
25.2;1. Experimental Tools to Map Forces and Kinematics during Collective Cell Migration;338
25.2.1;1.1 Polyacrylamide Gel Preparation;339
25.2.1.1;1.1.1 Equipment and reagents;339
25.2.1.2;1.1.2 Protocol;339
25.2.1.2.1;1.1.2.1 Glass-bottom petri dish activation;339
25.2.1.2.2;1.1.2.2 Preparation of polyacrylamide gels;339
25.2.1.2.3;1.1.2.3 Gel functionalization;340
25.2.1.2.4;1.1.2.4 Collagen coating;340
25.2.2;1.2 Direct Cell Seeding;340
25.2.3;1.3 PDMS Membrane Barrier Assay;340
25.2.3.1;1.3.1 Reagents and materials;342
25.2.3.2;1.3.2 Clean room equipment;342
25.2.3.3;1.3.3 Protocol;343
25.2.3.3.1;1.3.3.1 SU-8 spinning on glass slides;343
25.2.3.3.2;1.3.3.2 SU-8 photolithography;343
25.2.3.3.3;1.3.3.3 SU-8 development and silanization;343
25.2.3.3.4;1.3.3.4 Fabrication of PDMS membranes;343
25.2.3.3.5;1.3.3.5 Membrane passivation and cell seeding;343
25.2.3.3.6;1.3.3.6 Membrane release;344
25.2.4;1.4 Magnetic PDMS Barrier Assay;344
25.2.4.1;1.4.1 Reagents and materials;344
25.2.4.2;1.4.2 Equipment;346
25.2.4.3;1.4.3 Protocol;346
25.2.4.3.1;1.4.3.1 Designing and printing the mold for the magnetic PDMS with the 3D printer;346
25.2.4.3.2;1.4.3.2 Fabrication of the magnetic PDMS stencil;346
25.2.4.3.3;1.4.3.3 Magnetic PDMS stencil sterilization, passivation, and cell seeding;346
25.3;2. Computational Tools to Map Forces and Kinematics during Collective Cell Migration;347
25.3.1;2.1 Image Acquisition;347
25.3.2;2.2 Image Registration;347
25.3.3;2.3 Computing Cell Velocities (PIV);348
25.3.4;2.4 Computing Cell Forces;350
25.3.4.1;2.4.1 Computation of gel displacements at the interface with the cell monolayer;350
25.3.4.2;2.4.2 Computation of cell tractions at the interface with the cell monolayer;352
25.3.4.2.1;2.4.2.1 Boussinesq algorithm for infinite gel substrate of finite thickness;352
25.3.4.2.2;2.4.2.2 Finite element method (FEM) for gel substrate of finite thickness;353
25.3.4.3;2.4.3 Computation of inter- and intra-cellular stresses: MSM;353
25.3.4.3.1;2.4.3.1 Formulation of the problem;353
25.3.4.3.2;2.4.3.2 Solution of the problem;354
25.4;General Conclusions;355
25.5;Acknowledgments;355
25.6;References;355
26;18. Practical aspects of the cellular force inference toolkit (CellFIT);358
26.1;Introduction;359
26.2;1. The Basic Steps in CellFIT;361
26.2.1;1.1 Image Segmentation;361
26.2.2;1.2 Mesh Generation;364
26.2.3;1.3 Angle Determination;365
26.2.4;1.4 Curvature Determination;367
26.2.5;1.5 Construct Young Equations;367
26.2.6;1.6 Assemble, Constrain, and Solve Tension Equations;369
26.2.7;1.7 Construct Laplace Equations;370
26.2.8;1.8 Assemble, Constrain, and Solve Pressure Equations;370
26.2.9;1.9 Display Results;371
26.2.10;1.10 Evaluate Solutions;373
26.3;2. Working with CellFIT Output;374
26.4;Acknowledgments;376
26.5;References;377
27;19. Quantification of collagen contraction in three-dimensional cell culture;380
27.1;Introduction;381
27.2;1. Method;382
27.2.1;1.1 Sample Preparation;382
27.2.1.1;1.1.1 Materials;384
27.2.1.1.1;1.1.1.1 Cell culture;384
27.2.1.1.2;1.1.1.2 Multicellular cancer cells spheroids;384
27.2.1.1.3;1.1.1.3 3D collagen assay;384
27.2.1.2;1.1.2 Method;384
27.2.1.2.1;1.1.2.1 Cell culturing;384
27.2.1.2.2;1.1.2.2 Multicellular cancer cells spheroids;384
27.2.1.2.3;1.1.2.3 3D collagen assay;385
27.3;2. Pseudo-speckle Microscopy;386
27.3.1;2.1 Microscopy;386
27.3.2;2.2 Basic Image Corrections;387
27.4;3. Software;387
27.4.1;3.1 General Parameters (Figure 3, Panel III.A);388
27.4.2;3.2 Parameters for Edge Detection (Figure 3, Panel III.B);388
27.4.3;3.3 Parameters for Cross-Correlation (Figure 3, Panel III.C);390
27.4.4;3.4 Interpolation and Filter Parameters (Figure 3, Panel III.D);392
27.4.5;3.5 Data and Image Recording and Data Structure;392
27.5;4. Data Analysis;394
27.6;5. Discussion;396
27.7;General Conclusion;397
27.8;Acknowledgments;397
27.9;References;398
28;20. Generation of biocompatible droplets for in vivo and in vitro measurement of cell-generated mechanical stresses;400
28.1;Introduction;401
28.2;1. Methods;402
28.2.1;1.1 Generation and Stabilization of Biocompatible Droplets;402
28.2.1.1;1.1.1 Materials;403
28.2.1.2;1.1.2 Equipment;403
28.2.1.3;1.1.3 Method;403
28.2.2;1.2 Functionalization of Droplets;406
28.2.2.1;1.2.1 Materials;406
28.2.2.2;1.2.2 Equipment;406
28.2.2.3;1.2.3 Method;406
28.2.3;1.3 Characterizing the Mechanical Properties of the Droplets;409
28.2.3.1;1.3.1 Materials;410
28.2.3.2;1.3.2 Equipment;410
28.2.3.3;1.3.3 Method;410
28.2.4;1.4 Use of Droplets in Different Applications;413
28.2.4.1;1.4.1 In vitro;413
28.2.4.1.1;1.4.1.1 Single cells and cells in 2D monolayers;413
28.2.4.1.2;1.4.1.2 Cell aggregates;413
28.2.4.2;1.4.2 Ex vivo and in vivo;413
28.3;2. Discussion;414
28.4;Conclusion;415
28.5;References;416
29;21. Laser induced wounding of the plasma membrane and methods to study the repair process;418
29.1;Introduction;419
29.2;1. Cell Deposition;420
29.2.1;1.1 Materials;420
29.2.1.1;1.1.1 Reagents;420
29.2.1.2;1.1.2 Equipments;420
29.2.2;1.2 Method;421
29.2.2.1;1.2.1 Cell keeping;421
29.2.2.2;1.2.2 Cell plating;421
29.3;2. Photodamage and Imaging;421
29.3.1;2.1 Materials;421
29.3.2;2.2 Equipments;422
29.3.3;2.3 Method;422
29.3.3.1;2.3.1 Chamber mounting;422
29.3.3.2;2.3.2 Cell imaging;423
29.3.3.3;2.3.3 UV laser calibration and cell damage;424
29.4;3. Following Plasma Membrane Damage and Repair;426
29.4.1;3.1 Materials;426
29.4.2;3.2 Method;426
29.5;4. Image Analysis;428
29.5.1;4.1 Software;428
29.5.2;4.2 Methods;429
29.6;5. Data Analysis;429
29.6.1;5.1 Software;429
29.6.2;5.2 Method;429
29.7;6. Discussion;432
29.7.1;6.1 Alternative methods to damage the plasma membrane;432
29.7.2;6.2 Plasma Membrane Repair Alteration;432
29.7.3;6.3 Troubleshooting;433
29.8;General Conclusions;433
29.9;References;433
30;22. Electrofusion of giant unilamellar vesicles to cells;436
30.1;Introduction;437
30.2;1. Preparation of GUVs by Electroformation;438
30.2.1;1.1 Materials;440
30.2.2;1.2 Equipment;440
30.2.3;1.3 Method;440
30.2.3.1;1.3.1 Preparation of lipid-coated, ITO-coated slides;440
30.2.3.2;1.3.2 Assembly of the electroformation chamber;440
30.3;2. Electrofusion of GUVs to Cells;441
30.3.1;2.1 Materials;442
30.3.2;2.2 Equipment;443
30.3.3;2.3 Method;443
30.3.3.1;2.3.1 Prepare cell culture;443
30.3.3.2;2.3.2 Replating cells;443
30.3.3.3;2.3.3 Electrofusion;444
30.4;3. Discussion;445
30.5;References;447
31;23. Measurement and manipulation of cell size parameters in fission yeast;450
31.1;Introduction;451
31.2;1. Measurement of Size Parameters of Single Fission Yeast Cells;452
31.2.1;1.1 Dynamic Measurement of Cell Size Parameters During Single Spore Growth and Polarization;452
31.2.1.1;1.1.1 Spore preparation for imaging;452
31.2.1.2;1.1.2 Imaging;453
31.2.1.3;1.1.3 Image analysis;453
31.2.2;1.2 Length, Diameter, Surface, and Volume of Dividing Cells;455
31.2.2.1;1.2.1 Cell preparation for imaging;456
31.2.2.2;1.2.2 Image analysis;456
31.3;2. Microchannel Assay for Cell Diameter Manipulation;457
31.3.1;2.1 Fabricating Microchannels to Manipulate Cell Diameter;457
31.3.1.1;2.1.1 Photomask design;459
31.3.1.2;2.1.2 Photolithography;459
31.3.1.3;2.1.3 Creating PDMS from master;460
31.3.1.4;2.1.4 Assembling the micro channels;460
31.3.2;2.2 Cell Diameter Manipulation and Imaging;461
31.4;Conclusions;462
31.5;Acknowledgments;462
31.6;References;462
32;24. Methods for rectifying cell motions in vitro: breaking symmetry using microfabrication and microfluidics;464
32.1;Introduction;465
32.2;Relevance of Cell Migration In vivo;465
32.3;Origin of Symmetry Breaking In vivo and the Need for Controlled In vitro Approaches: Microfabrication and Microfluidics;466
32.4;1. Breaking Symmetry with Topography: Fabrication of a Topographical Pattern;466
32.4.1;1.1 Topographical Pattern Design;466
32.4.1.1;1.1.1 Materials;467
32.4.1.2;1.1.2 Pattern design;468
32.4.2;1.2 Fabrication of the Micropatterned Substrate;468
32.4.2.1;1.2.1 Materials;468
32.4.2.2;1.2.2 Equipment;469
32.4.2.3;1.2.3 Method;469
32.5;2. Breaking Symmetry with Chemical Gradient: Preparation of the Fibronectin Gradient;471
32.5.1;2.1 Microfluidic Chip Design;471
32.5.1.1;2.1.1 Materials;471
32.5.1.2;2.1.2 Pattern design;471
32.5.2;2.2 Fabrication of the Microfluidic Chip;471
32.5.2.1;2.2.1 Materials;471
32.5.2.2;2.2.2 Equipment;471
32.5.2.3;2.2.3 Method;472
32.5.3;2.3 Fibronectin Gradient Formation;472
32.5.3.1;2.3.1 Materials;472
32.5.3.2;2.3.2 Equipment;473
32.5.3.3;2.3.3 Method;473
32.5.3.3.1;2.3.3.1 Surface activation;473
32.5.3.3.2;2.3.3.2 Gradient formation;473
32.6;3. Cell Migration Experiments;474
32.6.1;3.1 Materials;476
32.6.2;3.2 Equipment;476
32.6.3;3.3 Method;476
32.7;4. Discussion;477
32.8;Conclusions;478
32.9;References;478
33;25. Analyzing bacterial movements on surfaces;480
33.1;Introduction;481
33.2;1. Preparing Bacterial Suspension;482
33.2.1;1.1 Materials;482
33.2.2;1.2 Method;483
33.3;2. Tracking Bacteria on Solid Surfaces;484
33.3.1;2.1 Materials;484
33.3.2;2.2 Coating Microscope Coverslips;485
33.3.3;2.3 Mount Coverslip for Microscopy;488
33.3.4;2.4 Imaging and Tracking the Motion of Single Bacteria;489
33.4;3. Tracking Bacteria on Cells;491
33.4.1;3.1 Materials;491
33.4.2;3.2 Cleaning and Sterilizing Coverslips;492
33.4.3;3.3 Prepare Coverslips with a Monolayer of Mammalian Cells;492
33.4.4;3.4 Mount Coverslip with Mammalian Cells for Microscopy;492
33.4.5;3.5 Imaging and Tracking the Motion of Single Bacteria;493
33.5;4. Discussion Points;494
33.5.1;4.1 Know Your Bacterium;494
33.5.2;4.2 Choose Your Time Frame;494
33.5.3;4.3 Complementary Techniques;494
33.6;Conclusions;495
33.7;References;495
34;26. Advances in single-cell experimental design made possible by automated imaging platforms with feedback through segmentation;498
34.1;Introduction;499
34.2;1. In vitro Experiments where Automation is Important;500
34.2.1;1.1 An Example of Host–Pathogen Interaction Phenotyping: Malaria Parasites and Red Blood Cells;500
34.2.2;1.2 Long-Term Live Imaging in Immune System Cells;501
34.3;2. Preparation of Cells Described in this Chapter;502
34.3.1;2.1 Malaria Culture;502
34.3.2;2.2 Macrophages;503
34.4;3. Automation Methods;503
34.4.1;3.1 Live Imaging Conditions and Microscope Setup;503
34.4.2;3.2 Development of Tracking Algorithm, Testing on Videos of Multiple Cell Types;503
34.4.3;3.3 Requirements of a Good Image Analysis Solution;504
34.4.4;3.4 A Specific Example of an Effective Segmentation Routine;505
34.4.5;3.5 Connecting Image Analysis to Microscope Hardware;505
34.4.6;3.6 The Automation Concept Deployed on Egress/Invasion, and Tweezers Intervention, in Malaria;508
34.5;4. Discussion;510
34.5.1;4.1 Potential Throughput of Single-Cell Tracking Experiments;510
34.5.2;4.2 Implications for Live Imaging;511
34.6;5. Outlook;512
34.7;Acknowledgments;514
34.8;References;514
35;Volumes in Series;516
36;Index;528

Leseproben

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...