Cellular Electron Microscopy, Vol. 79 Book

Contributors     xv
Introduction     xxi
Preparing Cells for Electron Microscopy
The Physics of Rapid Cooling and Its Implications for Cryoimmobilization of Cells   Jacques Dubochet
Introduction     8
Freezing Water     9
Biological Material     12
Freezing Biological Material     13
References     20
Cryopreparation Methods for Electron Microscopy of Selected Model Systems   Kent McDonald
Introduction     24
Equipment and Materials     26
General Rules for Loading Samples for HPF     31
Methods for Specific Organisms     34
Postfreezing Processing     38
Summary     52
References     52
Cryopreparation Methodology for Plant Cell Biology   Michael W. Hess
Introduction     58
Rationale     59
Methods     63
Materials     68
Discussion     68
Concluding Remarks     90
References     91
Correlative Light and Electron Microscopy of Early Caenorhabditis elegans Embryos in Mitosis   Thomas Muller-Reichert   Martin Srayko   Anthony Hyman   Eileen T. O'Toole   KentMcDonald
Introduction     102
Rationale     104
Methods     105
Instrumentation and Materials     111
Discussion     112
References     117
Imaging Fixed Cells in Three Dimensions
Understanding Microtubule Organizing Centers by Comparing Mutant and Wild-Type Structures with Electron Tomography   Eileen T. O'Toole   Thomas H. Giddings, Jr.   Susan K. Dutcher
Introduction     126
Rationale     127
Methods     128
Instrumentation and Materials     138
Discussion     139
Summary     141
References     141
Whole-Cell Investigation of Microtubule Cytoskeleton Architecture by Electron Tomography   J. L. Hoog   C. Antony
Introduction     146
Rationale     147
Methods     147
Materials     161
Discussion     163
Summary     165
References     165
Electron Microscopy of Archaea   Charles E. Robertson
Introduction     170
Rationale     172
Methods and Materials     179
Discussion     189
Summary     189
References      190
Reconstructing Mammalian Membrane Architecture by Large Area Cellular Tomography   Brad J. Marsh
Introduction and Rationale     194
Methods and Materials     196
Discussion     211
Summary     215
References     216
Visualization of Membrane-Cytoskeletal Interactions During Plant Cytokinesis   Marisa S. Otegui   Jotham R. Austin, II
Introduction     222
Rationale     223
Materials and Methods     224
Discussion     237
Summary     238
References     239
Electron Tomographic Methods for Studying the Chemical Synapse   Christopher P. Arthur   David B. Serrell   Maria Pagratis   David L. Potter   Dudley S. Finch   Michael H. B. Stowell
Introduction and Rationale     242
Neurons, Neural Networks, and the Synapse     242
ET and the Synapse     243
Types of Neurons and Synapses     244
Sources of Neurons for Structural Studies     246
Methods for Assessing Neuron and Synapse Integrity     247
Rapid Freezing and HPF of Neurons     248
Modeling and Analysis of Synaptic Structures     249
Stimulation-Dependent Changes in Synaptic Structure     252
The Future of EM Tomography and Synapses     252
Conclusions     255
References     255
Using Electron Microscopy to Understand Functional Mechanisms of Chromosome Alignment on the Mitotic Spindle   Bruce F. McEwen   Yimin Dong   Kristin J. VandenBeldt
Introduction     260
Basic Methodology     263
Organization of MTs in the Mitotic Spindle     267
Deciphering Mechanisms of Chromosome Alignment and K-Fiber Formation Using Correlative Microscopy     272
Toward a Molecular Understanding of Kinetochore Architecture     276
Beyond Morphology: Use of Electron Tomography to Model Kinetochore Control of MT Dynamics     283
Conclusions and Future Directions     287
References     287
Electron Microscopic Analysis of the Leading Edge in Migrating Cells   Tatyana Svitkina
Introduction     296
Understanding the Mechanisms of Leading Edge Protrusion: Complementary Roles of LM and EM     297
Platinum Replica EM of the Leading Edge Cytoskeleton     301
Identification of Cytoskeletal Components in Platinum Replica Preparations     315
References     316
Imaging Actomyosin In Situ   Kenneth A. Taylor   Shenping Wu   Mary C. Reedy   Michael K. Reedy
Introduction and Overview     322
Preparing the Specimen     327
Data Collection and Tomogram Calculation     334
Identifying Similar Structures in an Ensemble     342
Building Atomic Models     355
Relating Class Averages to the Specimen     358
Application to IFM     360
Application to Other Types of Specimen     361
Summary     362
References     363
Imaging Frozen-Hydrated Cells and Cell Parts
Electron Tomography of Bacterial Chemotaxis Receptor Assemblies   Peijun Zhang   Robert M. Weis   Peter J. Peters   Sriram Subramaniam
Introduction     374
Analysis of Tsr Assemblies in Membrane Extracts     375
Electron Tomography of Fixed, Cryosectioned E. coli Cells     377
Cryo-Electron Microscopy of Frozen-Hydrated Sections of E. coli Cells     378
Structure of Chemotaxis Receptors Using Cryo-Electron Tomography     381
Summary     383
References     384
How to "Read" a Vitreous Section   Jacques Dubochet   Benoit Zuber   Mikhail Eltsov   Cedric Bouchet-Marquis   Ashraf Al-Amoudi   Francoise Livolant
Introduction     386
Conventional Versus Cryo-Electron Microscopy: A Comparison     387
Illustration: Two Pitfalls     400
References     403
Single-Particle Electron Cryomicroscopy of the Ion Channels in the Excitation-Contraction Coupling Junction   Irina I. Serysheva   Wah Chiu   Steven J. Ludtke
Introduction     408
Single-Particle Cryo-EM Methodology     411
3D Reconstruction of Proteins in Triad Junction     418
Spatial Arrangement of DHPRs and RyRs in Triad Junctions     428
Summary     429
References     430
Electron Microscopy of Microtubule-Based Cytoskeletal Machinery   Andreas Hoenger   Daniela Nicastro
Introduction     439
Rationale     442
Methods     443
Discussion     457
References     458
Reconstructing the Endocytotic Machinery   Yifan Cheng   Thomas Walz
Introduction     464
Rationale     465
Methods     467
Results     470
Discussion     482
Summary     484
References     485
Localizing Macromolecules in Cells
3D Immunolocalization with Plastic Sections   Mary K. Morphew
Introduction     494
Rationale for the Various Approaches to Immuno-EM     495
Methods for Immuno-EM     496
Comparison of the Methods     504
Summary     511
References     511
Electron Microscopy Analysis of Viral Morphogenesis   Annegret Pelchen-Matthews   Mark Marsh
Introduction     516
Analysis of Virus Assembly on Plastic Sections     517
Immunolabeling of Ultrathin Cryosections: Applications of the Tokuyasu Technique to Study Virus Assembly     524
Future Developments     537
Summary and Conclusions     538
References     539
Electron Tomography of Immunolabeled Cryosections   Mark S. Ladinsky   Kathryn E. Howell
Introduction     544
Methods     545
Results and Discussion     549
References     557
Visualizing Macromolecules with Fluoronanogold: From Photon Microscopy to Electron Tomography   T. Cheutin   C. Sauvage   P. Tchelidze   M. F. O'Donohue   H. Kaplan   A. Beorchia   D. Ploton
Introduction     560
Experimental Approach for Immunostaining of pKi-67 and RNAP I      560
Applications     562
Discussion     571
References     573
Markers for Correlated Light and Electron Microscopy   Gina E. Sosinsky   Ben N. G. Giepmans   Thomas J. Deerinck   Guido M. Gaietta   Mark H. Ellisman
Introduction     576
How Do LM and EM Complement Each Other?     576
Fluorescence Photooxidation     577
Enzymatic-Based Methods     583
Particle-Based Methods for Protein Localization     584
Concluding Remarks     587
References     588
Localizing Specific Elements Bound to Macromolecules by EFTEM   Richard D. Leapman   Maria A. Aronova
Introduction     594
General Principles     595
Applications     604
Summary and Future Directions     611
References     612
Localization of Protein Complexes by Pattern Recognition   Christoph Best   Stephan Nickell   Wolfgang Baumeister
Introduction     616
Template Matching     616
The Missing-Wedge Problem     631
Applications     633
Conclusions     635
References     637
Aspects of Data Collection and Analysis
The Application of Energy-Filtered Electron Microscopy to Tomography of Thick, Selectively Stained Biological Samples   James C. Bouwer   Mason R. Mackey   Albert Lawrence   Tom J. Deerinck   Ying Z. Jones   Masako Terada   Maryann E. Martone   Steven T. Peltier   Mark H. Ellisman
Introduction     644
Introduction to MPL Imaging     646
Detailed Description of MPL Tomography     648
Techniques     651
Results     653
Conclusions     657
References     658
Optimization of Image Collection for Cellular Electron Microscopy   Paul Mooney
Introduction: Assessing the Role of Image Collection in Cellular Electron Microscopy     662
Applications: The Importance and Meaning of SSNR in Cell Biology Applications     665
Optimization of Detectors and of the Detection Process for Maximization of SSNR     675
Detectors: Proven Technologies     698
Detectors: Experimental Technologies     701
Discussion     714
Conclusions     715
References     716
Future Directions for Camera Systems in Electron Microscopy   Nguyen-Huu Xuong   Liang Jin   Stuart Kleinfelder   Shengdong Li   Philippe Leblanc   Fred Duttweiler   James C. Bouwer   Steven T. Peltier   Anna-Clare Milazzo   Mark Ellisman
Introduction     722
Background and Rationale     727
Description of the Direct Detection Detector     729
Detector Characterization     730
Radiation Damage     734
Discussion     736
References     737
Structure Determination In Situ by Averaging of Tomograms   Friedrich Forster   Reiner Hegerl
Introduction     742
Tomography and Its Increase in Resolution by Averaging     745
Coherent Averaging of Macromolecules in Practice     750
Applications of Tomogram Averaging     757
Conclusion and Outlook     760
References     763
Methods for Image Segmentation in Cellular Tomography   Kristian Sandberg
Introduction     770
Problem Formulation     771
Challenges in Cellular Tomography Segmentation     773
Important Considerations for Segmentation Methods     776
How Do We Know if the Segmentation Method Is Reliable?     778
Segmentation Methods for Cellular Tomography     781
Orientation-Based Segmentation      790
Results Using Orientation-Based Segmentation     793
Summary     797
References     797
Database Resources for Cellular Electron Microscopy   Maryann E. Martone   Joy Sargis   Joshua Tran   Willy Waiho Wong   Heather Jiles   Cem Mangir
Introduction     800
The Cell Centered Database Project     807
Looking Ahead     819
References     820
Index     823
Volumes in Series     843