Contributors ................................................. xiii
Preface ....................................................... xix
Volumes in Series ............................................. xxi
Section I. Organelles and Cellular Structure .................... 1
1 Monitoring Cytoskeletal Dynamics in Living Neurons Using
Fluorescence Photoactivation ................................. 3
Anne Gauthier-Kemper, Carina Weissmann, Hans-Jürgen
Reyher, and Roland Brandt
1 Introduction .............................................. 4
2 Generation and Expression of PAGFP Fusion Constructs
with Cytoskeletal Proteins ................................ 5
3 Fluorescence Photoactivation, Live Cell Imaging, and
Data Analysis ............................................ 12
Acknowledgments ............................................. 20
References .................................................. 20
2 Red/Far-Red Fluorescing DNA-Specific Anthraquinones for
Nucl:Cyto Segmentation and Viability Reporting in Cell-
Based Assays ................................................ 23
Roy Edward
1 Introduction ............................................. 24
2 The Basic Requirement for Counterstaining ................ 25
3 An Optimized Counterstain for GFP-Based Assays ........... 27
4 Cellular Compartment Segmentation ........................ 28
5 Labeling Cells in Live and Fixed End-Point Assays ........ 29
6 Protocol for Differential Dual Nuclear/Cytoplasmic
Counterstaining in Live Cells ............................ 31
7 Protocols for Differential Dual Nuclear/Cytoplasmic
Counterstaining in Fixed Cells ........................... 32
8 Measuring Cellular DNA Content ........................... 33
9 Protocol: DRAQ5TM DNA Content Measurement by Imaging ..... 34
10 Protocol: Cell Enumeration in "In-Cell Westerns" (After
Hannoush, 2008) .......................................... 36
11 Further Augmentation of Compartment Segmentation ......... 37
12 Cell Enumeration and Morphometries: Early Sentinels of
In Vitro Toxicology ......................................... 37
13 A Far-Red Viability Dye Derivative ....................... 39
14 Protocols: Use of DRAQ7TM to Identify Membrane-
Compromised Cells ........................................ 40
15 Summary .................................................. 42
Acknowledgment .............................................. 43
References .................................................. 43
3 Methods to Measure Actin Treadmilling Rate in Dendritic
Spines ...................................................... 47
Mikko Koskinen, Enni Bertling, and Pirta Hotulainen
1 Introduction ............................................. 48
2 Methods .................................................. 51
Acknowledgments ............................................. 57
References .................................................. 57
4 Live-Cell Imaging of Clathrin Coats ......................... 59
Comert Kural and Tom Kirchhausen
1 Introduction ............................................. 60
2 Dynamics of Clathrin Assembly ............................ 61
3 Limits of Fluorescence Microscopy ........................ 62
4 TIRF Microscopy .......................................... 63
5 How to Optimize Your TIRF System ......................... 64
6 Spinning-Disk Confocal Microscopy ........................ 65
7 Spherical Aberration Correction Applied to Spinning-
Disk Confocal Microscopy ................................. 66
8 Getting Around the Diffraction Limit ..................... 67
9 Use of 2D Spinning-Disk Confocal Microscope to Study
Clathrin-Mediated Endocytosis at the Ventral and Dorsal
Surfaces of a Cell ....................................... 69
10 Use of TIRF Microscopy to Study Clathrin-Mediated
Endocytosis .............................................. 70
11 The Third Dimension ...................................... 71
12 3D Tracking in Spinning-Disk Imaging ..................... 72
13 Use of 3D Tracking to Monitor Clathrin-Mediated Entry
of Reovirus Particles at the Apical Surface of
Polarized Cells .......................................... 74
14 Using the Optimum Pixel and Step Sizes in z-Stacks ....... 76
15 Conclusion ............................................... 76
Acknowledgments ............................................. 77
References .................................................. 77
5 Imaging the Mitotic Spindle ................................. 81
Paul S. Maddox, Anne-Marie Ladouceur, Rajesh Ranjan, Jonas
Dorn, Hery Ratsima, Damien D'Amours, and Amy S. Maddox
1 Introduction ............................................. 82
2 Modern Light Microscopy .................................. 83
3 Examples ................................................. 93
4 Conclusions ............................................. 101
Acknowledgments ............................................ 101
References ................................................. 101
6 Modeling of Spatial Intracellular Signaling Events in
Neurons .................................................... 105
Wendy C. Wenderski and Susana R. Neves
1 Introduction ............................................ 105
2 Computational Modeling of Intracellular Signaling ....... 106
3 Imaging Signaling Events in Striatal Neuronal Cultures .. 112
4 Experimental Validation of Model Predictions ............ 121
Acknowledgments ............................................ 121
References ................................................. 121
7 Imaging Chromosome Dynamics in Meiosis in Plants ........... 125
Moira J. Sheehan and Wojciech P. Pawіowski
1 Introduction ............................................ 126
2 Technical Considerations ................................ 128
3 Protocols and Methods ................................... 134
Acknowledgments ............................................ 141
References ................................................. 141
8 Analyzing Lysosomes in Live Cells .......................... 145
Paul R. Pryor
1 Introduction ............................................ 145
2 Visualizing Lysosomes ................................... 146
3 Measuring Lysosomal Ions ................................ 152
4 Delivery of Phagocytic Material to the Lysosome ......... 153
5 Endocytosis of Membrane Probes to the Lysosome .......... 154
References ................................................. 156
9 Detection and Quantification of Biomolecular Association
in Living Cells using Single-Molecule Microscopy ........... 159
Mario Brameshuber and Gerhard J. Schütz
1 Movements and Interactions Drive Biomolecular Function .. 160
2 The Concept: "Please Move to the Dark, so That We Can
See You" ................................................ 162
3 Proofing the Principle .................................. 164
4 Some Examples ........................................... 166
5 A User Guide to TOCCSL .................................. 176
6 Analysis ................................................ 179
7 Conclusions ............................................. 181
Acknowledgments ............................................ 182
References ................................................. 182
10 Visualizing Cardiac Ion Channel Trafficking Pathways ....... 187
James W. Smyth and Robin M. Shaw
1 Introduction ............................................ 188
2 Introducing Recombinant DNA for Ectopic Expression of
Fluorescent Ion Channel Fusion Proteins ................. 190
3 Isolating De Novo Ion Channel Forward Trafficking ....... 191
4 Recreating Cardiomyocyte-Like Membrane Subdomains in
Model Cell Systems ...................................... 192
5 Tools for Visualizing Forward Trafficking and Delivery
of Ion Channels in Real Time ............................ 197
6 Data Analysis ........................................... 201
7 Concluding Remarks ...................................... 201
Acknowledgments ............................................ 202
References ................................................. 202
11 Live Cell Imaging of the Cytoskeleton ...................... 203
Eve G. Stringham, Nancy Marcus-Gueret, Laura Ramsay, and
Kristopher L. Schmidt
1 Introduction ............................................ 204
2 Live Imaging of MTs in Tissue Cultured Cells ............ 206
3 Live Imaging of MTs in Whole Organisms .................. 209
4 Live Imaging of Actin Filaments in C. elegans ........... 211
References ................................................. 214
12 Fluorescence Single-Molecule Imaging of Actin Turnover
and Regulatory Mechanisms .................................. 219
Naoki Watanabe
1 Introduction ............................................ 220
2 Introducing a Low Density of Fluorescent Proteins in
XTC Cells ............................................... 222
3 Observation of XTC Cells Spreading on Poly-L-lysine
(PLL)-coated Glass Coverslips ........................... 224
4 Data Analysis ........................................... 229
Acknowledgments ............................................ 232
References ................................................. 232
13 Analysis of Cell Dispersion and Migration by Video-
Microscopy ................................................. 233
Bonnomet Arnaud, Terryn Christine, Cutrona Jérôme,
Jonquet Antoine, Birembaut Philippe, and Zahm Jean-Marie
1 Introduction ............................................ 234
2 Video-Microscope ........................................ 235
3 Model of Cell Dispersion ................................ 236
4 Models of Cell Migration in Sheet ....................... 238
5 Model of 3D Cell Migration .............................. 243
6 Quantification .......................................... 245
7 Conclusions ............................................. 252
References ................................................. 252
Section II. Molecules and Ions ................................ 255
14 Real Time Qualitative and Quantitative GLUT4
Translocation Assay ........................................ 257
Maleppillil Vavachan Vijayakumar and Manoj Kumar Bhat
1 Introduction ............................................ 258
2 GLUT4 Translocation Assay ............................... 259
3 Methods and Procedures .................................. 261
4 Conclusions and Future Applications ..................... 270
Acknowledgements ........................................... 270
References ................................................. 270
15 Advanced Imaging of Cellular Signaling Events .............. 273
Marek Cebecauer, Jana Humpolíčková, and Jerémie Rossy
1 Introduction ............................................ 274
2 Selection of Tools and Transfection of Cells ............ 275
3 Imaging of Cell Signaling Events ........................ 276
4 Data Analysis ........................................... 281
5 Summary ................................................. 287
Acknowledgments ............................................ 288
References ................................................. 288
16 Dynamic Imaging of Homo-FRET in Live Cells by
Fluorescence Anisotropy Microscopy ......................... 291
Subhasri Ghosh, Suvrajit Saha, Debanjan Goswami, Sameera
Bilgrami, and Satyajit Mayor
1 Introduction ............................................ 292
2 Theoretical Concepts .................................... 293
3 Homo-FRET Measurements in an Imaging Modality:
Practical Considerations ................................ 298
4 Implementation of High-Resolution Steady-State
Anisotropy Imaging Modalities ........................... 309
5 Detecting Homo-FRET Using Time Resolved Anisotropy
(TRA) Modalities ........................................ 319
6 Prospects and Perspectives .............................. 325
Acknowledgments ............................................ 325
References ................................................. 325
17 Time-Resolved Luminescence Resonance Energy Transfer
Imaging of Protein-Protein Interactions in Living Cells .... 329
Harsha E. Rajapakse and Lawrence W. Miller
1 Introduction ............................................ 330
2 Lanthanide Protein Labels ............................... 332
3 Transfection and Cellular Delivery of TMP-Lumi4 ......... 335
4 Time-Resolved LRET Microscopy ........................... 338
Acknowledgment ............................................. 344
References ................................................. 344
18 Imaging of Corticosteroid Receptors in Live Cells .......... 347
Mayumi Nishi
1 Introduction ............................................ 348
2 Time-Lapse Imaging of GR and MR in a Single Cell ........ 349
3 Subcellular and Intranuclear Dynamics of GR and MR
Examined by FRAP Analyses ............................... 354
4 Interaction of Corticosteroid Receptors in the Nucleus .. 354
5 Notes ................................................... 357
Acknowledgment ............................................. 360
References ................................................. 360
19 Investigating Second Messenger Signaling In Vivo ........... 363
Rüdiger Rudolf, Mathias Hafner, and Marco Mongillo
1 Introduction ............................................ 364
2 Multimodal Imaging in Living Mouse Skeletal Muscle ...... 365
3 Adaptations to Tissues Other Than Skeletal Muscle ....... 375
4 Concluding Remarks ...................................... 380
Acknowledgments ............................................ 380
References ................................................. 380
20 Probes for Intracellular RNA Imaging in Live Cells ......... 383
Philip J. Santangelo, Eric Alonas, Jeenah Jung, Aaron
W. Lifland, and Chiara Zurla
1 Introduction ............................................ 384
2 Imaging RNA in Live Cells ............................... 385
3 Limitations of Single-Label Probes and Molecular
Beacons ................................................. 386
4 Imaging Native, Nonengineered RNA in Live Cells with
Single RNA Sensitivity Using Exogenous Probes ........... 389
5 Tпrne-Lapse Imaging of Native, Nonengineered β-actin
mRNA Granule Dynamics ................................... 394
6 Imaging RNA-Protein Colocalization Using MTRIPs ......... 395
7 Conclusions ............................................. 396
References ................................................. 397
21 Imaging the Glycome in Living Systems ...................... 401
Boyangzi Li, Feiyan Mock, and Peng Wu
1 Introduction ............................................ 402
2 Bioorthogonal Chemistry in Glycan Labeling: Merits and
Limitations ............................................. 403
3 Metabolic Labeling of Cell Surface Fucosylated Glycans
in Zebrafish Embryos for Fluorescence Imaging ........... 406
4 Labeling Cell Surface Glycans Bearing the LacNAc
Disaccharide in Chinese Hamster Ovary(CHO) Cells for
Fluorescence Imaging .................................... 412
Acknowledgments ............................................ 416
References ................................................. 416
22 Intracellular Magnesium Detection by Fluorescent
Indicators ................................................. 421
Valentina Trapani, Monika Schweigel-Röntgen, Achille
Cittadini, and Federica I. Wolf
1 Introduction ............................................ 422
2 Fluorescent Indicators for Magnesium .................... 424
3 Magnesium Detection in Live Cells by
Spectrofluorimetry ...................................... 428
4 Magnesium Detection in Live Cells by Microscopy ......... 434
5 Future Perspectives ..................................... 441
Acknowledgments ............................................ 442
References ................................................. 442
23 Illuminating Mobile Zinc with Fluorescence: From Cuvettes
to Live Cells and Tissues .................................. 445
Zhen Huang and Stephen J. Lippard
1 Introduction ............................................ 446
2 A Guide to Fluorescent Zinc Probes ...................... 448
3 Representative Procedure for Imaging Mobile Zinc in
Live Cells .............................................. 457
4 Imaging Endogenous Neuronal Zinc in Live Cells and
Tissues ................................................. 462
5 Concluding Remarks ...................................... 464
Acknowledgments ............................................ 465
References ................................................. 465
24 Quantitative Fluorescent Live Cell Imaging of
Intracellular Ca2+ and H+ Ions in Malaria Parasites ........ 469
Petra Rohrbach
1 Introduction ............................................ 470
2 Live Cell Imaging of Intact Malaria Parasites ........... 471
3 Measuring Ion Homeostasis in P. falciparum Using
Fluorescent .............................................
Indicators ................................................. 492
Acknowledgment ............................................. 492
References ................................................. 485
Author Index .................................................. 485
Subject Index.................................................. 511
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