Contributors ................................................... xi
Preface ...................................................... xvii
Volumes in Series ............................................. xix
Section I. Techniques ........................................... 1
1 Laser-Induced Radiation Microbeam Technology and
Simultaneous Real-Time Fluorescence Imaging in Live Cells .... 3
Stanley W. Botchway, Pamela Reynolds, Anthony W. Parker,
and Peter O'Neill
1 Introduction .............................................. 4
2 Experimental Procedure ................................... 10
3 Conclusion/Forward Look .................................. 25
Acknowledgments ............................................. 26
References .................................................. 26
2 A Cell Biologist's Guide to High Resolution Imaging ......... 29
Graeme Ball, Richard M. Parton, Russell S. Hamilton, and
Ian Davis
1 Introduction ............................................. 30
2 Physical Limitations on the Resolution of Conventional
Microscopy ............................................... 31
3 Preparations for High Resolution Fluorescence Imaging .... 35
4 High Resolution Image Data Acquisition and Processing .... 39
5 Processing ............................................... 43
6 Analysis of High Resolution Image Data ................... 47
7 Conclusions .............................................. 51
Acknowledgments ............................................. 51
References .................................................. 51
3 Applications of Fluorescence Lifetime Spectroscopy and
Imaging to Lipid Domains In Vivo ............................ 57
André E.P. Bastos, Silvia Scolari, Martin Stöckl, and
Rodrigo F.M. de Almeida
1 The Challenge of Studying Lipid Domains In Vivo Defied
by Fluorescence Lifetimes ................................ 58
2 Importance of Studies In Vitro to Design and
Rationalize Studies In Vivo .............................. 61
3 Labeling Cell Membranes In Vivo: Lipophilic Probes
Versus GFP-Tagged Membrane Proteins ...................... 67
4 Cuvette Lifetimes and FLIM: In Vivo Applications ......... 70
Acknowledgments ............................................. 78
References .................................................. 79
4 Detecting and Tracking Nonfluorescent Nanoparticle Probes
in Live Cells .............................................. 83
Gufeng Wang and Ning Fang
1 Introduction ............................................. 84
2 Techniques and Tools ..................................... 84
3 Biological Applications .................................. 90
4 Cytotoxicity of Nanoparticle Probes ..................... 100
5 Conclusions and Future Perspective ...................... 101
Acknowledgments ............................................ 102
References ................................................. 102
5 Fluorescence Lifetime Microscopy of Tumor Cell Invasion,
Drug Delivery, and Cytotoxicity ............................ 109
Gert-jan Bakker, Volker Andresen, Robert M. Hoffman, and
Peter Friedl
1 Introduction ............................................ 110
2 Preparation of Mouse Mammary Tumor Cell Spheroids in a
Collagen Matrix ......................................... 112
3 Simultaneous Acquisition of Fluorescence Lifetime and
Intensity to Monitor DOX Uptake ......................... 113
4 Monitoring Drug Uptake Kinetics by FLIM ................. 119
5 Conclusions and Outlook ................................. 123
Acknowledgment ............................................. 123
References ................................................. 124
6 Measuring Membrane Protein Dynamics in Neurons Using
Fluorescence Recovery after Photobleach .................... 127
Inmaculada M. González-González, Frédéric Jaskуlski,
Yves Goldberg, Michael C. Ashby, and Jeremy M. Henley
1 Introduction ............................................ 128
2 Experimental Parameters and Preparation ................. 130
3 Equipment ............................................... 133
4 Establishing FRAP Conditions ............................ 134
5 A Basic FRAP Protocol ................................... 136
6 Analysis of FRAP ........................................ 137
7 Acknowledgments References
Fluorescent Speckle Microscopy in Cultured Cells ........... 147
Marin Barisic, António J. Pereira, and Helder Maiato
1 Introduction ............................................ 148
2 Technical Challenges of FSM ............................. 151
3 Detailed Methodology for FSM in Cultured Cells .......... 153
4 Future Challenges Acknowledgments ....................... 159
References ................................................. 159
8 Green-to-Red Photoconvertible mEosFP-Aided Live Imaging in
Plants ..................................................... 164
Jaideep Mathur, Sarah Griffiths, Kiah Barton, and
Martin H. Schattat
1 Introduction ............................................ 164
2 Expression of mEosFP Fusion Proteins in Plants .......... 167
3 Visualization of mEosFP Probes in Plants ................ 170
4 Uses of mEosFP Probes in Plants ......................... 173
5 Post Acquisition Image Processing and Data Creation ..... 177
Acknowledgments ............................................ 178
References ................................................. 179
9 Methods for Cell and Particle Tracking ..................... 183
Erik Meijering, Oleh Dzyubachyk, and lhor Smal
1 Introduction ............................................ 184
2 Tracking Approaches ..................................... 185
3 Tracking Tools .......................................... 187
4 Tracking Measures ....................................... 192
5 Tips and Tricks ......................................... 195
Acknowledgments ............................................ 197
References ................................................. 197
10 Correlative Light-Electron Microscopy: A Potent Tool
for the Imaging of Rare or Unique Cellular and Tissue
Events and Structures ...................................... 201
Alexander A. Mironov and Galina V. Beznoussenko
1 Introduction ............................................ 202
2 Observation of Living Cells and Fixation ................ 203
3 Immunolabeling with NANOGOLD ............................ 207
4 Enhancement of Sample Contrast, Sample Locating, and
Embedding
5 Identification of the Cell of Interest on EPON Blocks ... 211
6 Sample Orientation and EM Sectioning from the Very
First Section ........................................... 212
7 Picking up Serial Sections with the Empty Slot Grid ..... 214
EM Analysis ................................................ 216
Acknowledgments ............................................ 217
References
11 Optical Techniques for Imaging Membrane Domains in Live
Cells (Live-Cell Palm of Protein Clustering) ............... 221
Dylan M. Owen, David Williamson, Astrid Magenau, and
Katharina Gaus
1 Introduction ............................................ 222
2 Sample Preparation and Data Acquisition ................. 224
3 Data Analysis ........................................... 228
Acknowledgments ............................................ 234
References ................................................. 234
12 Single Live Cell Topography and Activity Imaging with the
Shear-Force-Based Constant-Distance Scanning
Electrochemical Microscope ................................. 237
Albert Schulte, Michaela Nebel, and Wolfgang Schuhmann
1 Introduction ............................................ 238
2 Shear-Force-Based Constant-Distance Scanning
Electrochemical Microscopy for Live Cell Studies:
Apparatus, Probes, and Operation ........................ 240
3 Selected Applications of SF-CD-SECM Live Cell Studies ... 248
Acknowledgments ............................................ 253
References ................................................. 254
13 Visualization of TGN-Endosome Trafficking in Mammalian
and Drosophila Cells ....................................... 255
Satoshi Kametaka and Satoshi Waguri
1 Introduction ............................................ 256
2 Molecular Tools ......................................... 258
3 Live-Cell Imaging in Mammalian Cells .................... 260
4 Live-Cell Imaging in Drosophila Cells ................... 266
5 Conclusion Remarks ...................................... 269
References ................................................. 269
14 Live Cell Imaging with Chemical Specificity Using Dual
Frequency CARS Microscopy .................................. 273
Lestyn Pope, Wolfgang Langbein, Paola Borri, and Peter
Watson
1 Introduction ............................................ 274
2 "Noninvasive" Live Cell Imaging ......................... 275
3 Experimental Setup ...................................... 279
4 Maximizing Collection Efficiency for Live Cell Imaging .. 287
Acknowledgments ............................................ 289
References ................................................. 290
15 Imaging Intracellular Protein Dynamics by Spinning Disk
Confocal Microscopy ........................................ 293
Samantha Stehbens, Hayley Pemble, Lyndsay Murrow, and
Torsten Wittmann
1 Introduction ............................................ 294
2 Instrument Design ....................................... 298
3 Combination with Other Imaging Techniques ............... 304
4 Specimen Preparation .................................... 307
Acknowledgments ............................................ 312
References ................................................. 312
Section II. Tools ............................................. 315
16 Visualizing Dynamic Activities of Signaling Enzymes Using
Genetically Encodable FRET-Based Biosensors: From Designs
to Applications ............................................ 317
Xin Zhou, Katie J. Herbst-Robinson, and Jin Zhang
1 Introduction ............................................ 318
2 Generalizable Modular Designs ........................... 319
3 FRET-Based Biosensors for Monitoring Signaling Enzymes .. 324
4 Example: A-kinase Activity Reporter (AKAR) .............. 332
5 Summary and Perspectives ................................ 336
Acknowledgments ............................................ 337
References ................................................. 337
17 Live-Cell Imaging of Aquaporin-4 Supramolecular Assembly
and Diffusion .............................................. 341
A.S. Verkman, Andrea Rossi, and Jonathan M. Crane
1 Aquaporin-4 (AQP4) and Orthogonal Arrays of Particles ... 342
2 Approaches to Image AQP4 and OAPs ....................... 343
3 AQP4 Diffusion and OAPs Studied by Quantum Dot Single
Particle Tracking ....................................... 345
4 OAP Dynamics and Structure Studied with GFP-AQP4
Chimeras ................................................ 347
5 Single-Molecule Analysis Shows AQP4 Heterotetramers ..... 349
6 Photobleaching Reveals Post-Golgi Assembly of OAPs ...... 351
7 Super-Resolution Imaging of AQP4 OAPs ................... 351
References ................................................. 352
18 Coiled-Coil Tag-Probe Labeling Methods for Live-Cell
Imaging of Membrane Receptors .............................. 355
Yoshiaki Yano, Kenichi Kawano, Kaoru Omae, and Katsumi
Matsuzaki
1 Introduction ............................................ 356
2 Various Principles Used for Tag-Probe Labeling .......... 357
3 Coiled-Coil Tag-Probe Labeling .......................... 360
4 Applications ............................................ 366
Acknowledgments ............................................ 368
References ................................................. 368
19 Monitoring Protein Interactions in Living Cells with
Fluorescence Lifetime Imaging Microscopy ................... 371
Yuansheng Sun, Nicole M. Hays, Ammasi Periasamy,
Michael W. Davidson, and Richard N. Day
1 Introduction ............................................ 372
2 FD FLIM Measurements .................................... 376
3 Measuring Protein-Protein Interactions in Living Cells
Using FLIM-FRET ......................................... 385
4 The Strengths and Limitations of FLIM ................... 388
Acknowledgment ............................................. 389
References ................................................. 389
20 Open Source Tools for Fluorescent Imaging .................. 393
Nicholas A. Hamilton
1 Why Open Source Software? ............................... 394
2 Open Source Software for Microscopy Imaging and
Analysis ................................................ 406
3 The Future .............................................. 414
Acknowledgments ............................................ 415
References ................................................. 415
21 Nanoparticle PEBBLE Sensors in Live Cells .................. 419
Yong-Eun Koo Lee and Raoul Kopelman
1 Introduction ............................................ 420
2 PEBBLE Sensor Designs ................................... 424
3 Preparation/Characterization ............................ 430
4 Examples ................................................ 431
5 Summary and Critical Issues ............................. 461
Acknowledgments ............................................ 462
References ................................................. 462
Author Index .................................................. 471
Subject Index ................................................. 495
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