Cellular Electron Microscopy

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ISBN: 9780123706478 | 0123706475
Cover: Hardcover
Copyright: 2/22/2007

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Recent improvements in electron microscopy of cells are bringing cell biologists back to "EM", particularly as they realize the limitations of the light microscopic methods that have been dominating the study of cell structure. This trend is a direct result of the resolving power of the electron microscope, of improved methods for specimen preparation, and of new technologies for three-dimensional imaging and image analysis. EM is beginning to fulfill its promise of linking light microscopy of live cells with atomic resolution studies of isolated macromolecules, with the goal of improving our understanding of living systems at atomic resolution. These new methods will help biologists from many disciplines to apply modern EM to their own work, allowing them to participate in the groundbreaking discoveries that are now being made in this field.

Contributors	p. xv
Introduction	p. xxi
Preparing Cells for Electron Microscopy
The Physics of Rapid Cooling and Its Implications for Cryoimmobilization of Cells
Introduction	p. 8
Freezing Water	p. 9
Biological Material	p. 12
Freezing Biological Material	p. 13
References	p. 20
Cryopreparation Methods for Electron Microscopy of Selected Model Systems
Introduction	p. 24
Equipment and Materials	p. 26
General Rules for Loading Samples for HPF	p. 31
Methods for Specific Organisms	p. 34
Postfreezing Processing	p. 38
Summary	p. 52
References	p. 52
Cryopreparation Methodology for Plant Cell Biology
Introduction	p. 58
Rationale	p. 59
Methods	p. 63
Materials	p. 68
Discussion	p. 68
Concluding Remarks	p. 90
References	p. 91
Correlative Light and Electron Microscopy of Early Caenorhabditis elegans Embryos in Mitosis
Introduction	p. 102
Rationale	p. 104
Methods	p. 105
Instrumentation and Materials	p. 111
Discussion	p. 112
References	p. 117
Imaging Fixed Cells in Three Dimensions
Understanding Microtubule Organizing Centers by Comparing Mutant and Wild-Type Structures with Electron Tomography
Introduction	p. 126
Rationale	p. 127
Methods	p. 128
Instrumentation and Materials	p. 138
Discussion	p. 139
Summary	p. 141
References	p. 141
Whole-Cell Investigation of Microtubule Cytoskeleton Architecture by Electron Tomography
Introduction	p. 146
Rationale	p. 147
Methods	p. 147
Materials	p. 161
Discussion	p. 163
Summary	p. 165
References	p. 165
Electron Microscopy of Archaea
Introduction	p. 170
Rationale	p. 172
Methods and Materials	p. 179
Discussion	p. 189
Summary	p. 189
References	p. 190
Reconstructing Mammalian Membrane Architecture by Large Area Cellular Tomography
Introduction and Rationale	p. 194
Methods and Materials	p. 196
Discussion	p. 211
Summary	p. 215
References	p. 216
Visualization of Membrane-Cytoskeletal Interactions During Plant Cytokinesis
Introduction	p. 222
Rationale	p. 223
Materials and Methods	p. 224
Discussion	p. 237
Summary	p. 238
References	p. 239
Electron Tomographic Methods for Studying the Chemical Synapse
Introduction and Rationale	p. 242
Neurons, Neural Networks, and the Synapse	p. 242
ET and the Synapse	p. 243
Types of Neurons and Synapses	p. 244
Sources of Neurons for Structural Studies	p. 246
Methods for Assessing Neuron and Synapse Integrity	p. 247
Rapid Freezing and HPF of Neurons	p. 248
Modeling and Analysis of Synaptic Structures	p. 249
Stimulation-Dependent Changes in Synaptic Structure	p. 252
The Future of EM Tomography and Synapses	p. 252
Conclusions	p. 255
References	p. 255
Using Electron Microscopy to Understand Functional Mechanisms of Chromosome Alignment on the Mitotic Spindle
Introduction	p. 260
Basic Methodology	p. 263
Organization of MTs in the Mitotic Spindle	p. 267
Deciphering Mechanisms of Chromosome Alignment and K-Fiber Formation Using Correlative Microscopy	p. 272
Toward a Molecular Understanding of Kinetochore Architecture	p. 276
Beyond Morphology: Use of Electron Tomography to Model Kinetochore Control of MT Dynamics	p. 283
Conclusions and Future Directions	p. 287
References	p. 287
Electron Microscopic Analysis of the Leading Edge in Migrating Cells
Introduction	p. 296
Understanding the Mechanisms of Leading Edge Protrusion: Complementary Roles of LM and EM	p. 297
Platinum Replica EM of the Leading Edge Cytoskeleton	p. 301
Identification of Cytoskeletal Components in Platinum Replica Preparations	p. 315
References	p. 316
Imaging Actomyosin In Situ
Introduction and Overview	p. 322
Preparing the Specimen	p. 327
Data Collection and Tomogram Calculation	p. 334
Identifying Similar Structures in an Ensemble	p. 342
Building Atomic Models	p. 355
Relating Class Averages to the Specimen	p. 358
Application to IFM	p. 360
Application to Other Types of Specimen	p. 361
Summary	p. 362
References	p. 363
Imaging Frozen-Hydrated Cells and Cell Parts
Electron Tomography of Bacterial Chemotaxis Receptor Assemblies
Introduction	p. 374
Analysis of Tsr Assemblies in Membrane Extracts	p. 375
Electron Tomography of Fixed, Cryosectioned E. coli Cells	p. 377
Cryo-Electron Microscopy of Frozen-Hydrated Sections of E. coli Cells	p. 378
Structure of Chemotaxis Receptors Using Cryo-Electron Tomography	p. 381
Summary	p. 383
References	p. 384
How to "Read" a Vitreous Section
Introduction	p. 386
Conventional Versus Cryo-Electron Microscopy: A Comparison	p. 387
Illustration: Two Pitfalls	p. 400
References	p. 403
Single-Particle Electron Cryomicroscopy of the Ion Channels in the Excitation-Contraction Coupling Junction
Introduction	p. 408
Single-Particle Cryo-EM Methodology	p. 411
3D Reconstruction of Proteins in Triad Junction	p. 418
Spatial Arrangement of DHPRs and RyRs in Triad Junctions	p. 428
Summary	p. 429
References	p. 430
Electron Microscopy of Microtubule-Based Cytoskeletal Machinery
Introduction	p. 439
Rationale	p. 442
Methods	p. 443
Discussion	p. 457
References	p. 458
Reconstructing the Endocytotic Machinery
Introduction	p. 464
Rationale	p. 465
Methods	p. 467
Results	p. 470
Discussion	p. 482
Summary	p. 484
References	p. 485
Localizing Macromolecules in Cells
3D Immunolocalization with Plastic Sections
Introduction	p. 494
Rationale for the Various Approaches to Immuno-EM	p. 495
Methods for Immuno-EM	p. 496
Comparison of the Methods	p. 504
Summary	p. 511
References	p. 511
Electron Microscopy Analysis of Viral Morphogenesis
Introduction	p. 516
Analysis of Virus Assembly on Plastic Sections	p. 517
Immunolabeling of Ultrathin Cryosections: Applications of the Tokuyasu Technique to Study Virus Assembly	p. 524
Future Developments	p. 537
Summary and Conclusions	p. 538
References	p. 539
Electron Tomography of Immunolabeled Cryosections
Introduction	p. 544
Methods	p. 545
Results and Discussion	p. 549
References	p. 557
Visualizing Macromolecules with Fluoronanogold: From Photon Microscopy to Electron Tomography
Introduction	p. 560
Experimental Approach for Immunostaining of pKi-67 and RNAP I	p. 560
Applications	p. 562
Discussion	p. 571
References	p. 573
Markers for Correlated Light and Electron Microscopy
Introduction	p. 576
How Do LM and EM Complement Each Other?	p. 576
Fluorescence Photooxidation	p. 577
Enzymatic-Based Methods	p. 583
Particle-Based Methods for Protein Localization	p. 584
Concluding Remarks	p. 587
References	p. 588
Localizing Specific Elements Bound to Macromolecules by EFTEM
Introduction	p. 594
General Principles	p. 595
Applications	p. 604
Summary and Future Directions	p. 611
References	p. 612
Localization of Protein Complexes by Pattern Recognition
Introduction	p. 616
Template Matching	p. 616
The Missing-Wedge Problem	p. 631
Applications	p. 633
Conclusions	p. 635
References	p. 637
Aspects of Data Collection and Analysis
The Application of Energy-Filtered Electron Microscopy to Tomography of Thick, Selectively Stained Biological Samples
Introduction	p. 644
Introduction to MPL Imaging	p. 646
Detailed Description of MPL Tomography	p. 648
Techniques	p. 651
Results	p. 653
Conclusions	p. 657
References	p. 658
Optimization of Image Collection for Cellular Electron Microscopy
Introduction: Assessing the Role of Image Collection in Cellular Electron Microscopy	p. 662
Applications: The Importance and Meaning of SSNR in Cell Biology Applications	p. 665
Optimization of Detectors and of the Detection Process for Maximization of SSNR	p. 675
Detectors: Proven Technologies	p. 698
Detectors: Experimental Technologies	p. 701
Discussion	p. 714
Conclusions	p. 715
References	p. 716
Future Directions for Camera Systems in Electron Microscopy
Introduction	p. 722
Background and Rationale	p. 727
Description of the Direct Detection Detector	p. 729
Detector Characterization	p. 730
Radiation Damage	p. 734
Discussion	p. 736
References	p. 737
Structure Determination In Situ by Averaging of Tomograms
Introduction	p. 742
Tomography and Its Increase in Resolution by Averaging	p. 745
Coherent Averaging of Macromolecules in Practice	p. 750
Applications of Tomogram Averaging	p. 757
Conclusion and Outlook	p. 760
References	p. 763
Methods for Image Segmentation in Cellular Tomography
Introduction	p. 770
Problem Formulation	p. 771
Challenges in Cellular Tomography Segmentation	p. 773
Important Considerations for Segmentation Methods	p. 776
How Do We Know if the Segmentation Method Is Reliable?	p. 778
Segmentation Methods for Cellular Tomography	p. 781
Orientation-Based Segmentation	p. 790
Results Using Orientation-Based Segmentation	p. 793
Summary	p. 797
References	p. 797
Database Resources for Cellular Electron Microscopy
Introduction	p. 800
The Cell Centered Database Project	p. 807
Looking Ahead	p. 819
References	p. 820
Index	p. 823
Volumes in Series	p. 843
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