Mcintosh Cellular Electron Microscopy

1. Auflage 2011
ISBN: 978-0-08-047503-5
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, Band Volume 79, 880 Seiten

Reihe: Methods in Cell Biology

Recent advances in the imaging technique electron microscopy (EM) have improved the method, making it more reliable and rewarding, particularly in its description of three-dimensional detail. Cellular Electron Microscopy will help biologists from many disciplines understand modern EM and the value it might bring to their own work. The book's five sections deal with all major issues in EM of cells: specimen preparation, imaging in 3-D, imaging and understanding frozen-hydrated samples, labeling macromolecules, and analyzing EM data. Each chapter was written by scientists who are among the best in their field, and some chapters provide multiple points of view on the issues they discuss. Each section of the book is preceded by an introduction, which should help newcomers understand the subject. The book shows why many biologists believe that modern EM will forge the link between light microscopy of live cells and atomic resolution studies of isolated macromolecules, helping us toward the goal of an atomic resolution understanding of living systems. - Updates the numerous technological innovations that have improved the capabilities of electron microscopy - Provides timely coverage of the subject given the significant rise in the number of biologists using light microscopy to answer their questions and the natural limitations of this kind of imaging - Chapters include a balance of 'how to', 'so what' and 'where next', providing the reader with both practical information, which is necessary to use these methods, and a sense of where the field is going

Autoren/Hrsg.

Mcintosh, J. Richard

Inhaltsverzeichnis

1;Front Cover;1
2;Methods in Cell Biology;4
3;Copyright Page;5
4;Contents;6
5;Contributors;16
6;Introduction;22
6.1;References;26
7;Part I: Preparing Cells for Electron Microscopy;29
7.1;References;32
7.2;Chapter 1: The Physics of Rapid Cooling and Its Implications for Cryoimmobilization of Cells;35
7.2.1;I. Introduction;36
7.2.2;II. Freezing Water;37
7.2.3;III. Biological Material;40
7.2.4;IV. Freezing Biological Material;41
7.2.4.1;A. Homogeneous and Heterogeneous Nucleation;42
7.2.4.2;B. Ice Formation in Biological Material;43
7.2.4.3;C. Vitrification;45
7.2.4.4;D. Devitrification;46
7.2.4.5;E. Recipe for Optimal Cryoimmobilization;47
7.2.5;Acknowledgments;48
7.2.6;References;48
7.3;Chapter 2: Cryopreparation Methods for Electron Microscopy of Selected Model Systems;51
7.3.1;I. Introduction;52
7.3.2;II. Equipment and Materials;54
7.3.2.1;A. High-Pressure Freezers;54
7.3.2.2;B. Specimen Carriers;55
7.3.2.3;C. Accessories;57
7.3.2.4;D. Cryoprotectants/Fillers;57
7.3.2.5;E. Tools Useful for Most HPF Specimen Loading Operations;59
7.3.3;III. General Rules for Loading Samples for HPF;59
7.3.3.1;A. Work Only with Healthy, Unstressed Cells;60
7.3.3.2;B. Work Quickly;60
7.3.3.3;C. Do Not Let Your Cells/Tissues Dry Out;60
7.3.3.4;D. Avoid Surrounding Your Cells/Organisms with Aqueous Media;61
7.3.3.5;E. Avoid Mechanical Damage;61
7.3.3.6;F. Fill the Carrier Correctly;61
7.3.3.7;G. Use the Smallest Volume of Sample You Can;61
7.3.4;IV. Methods for Specific Organisms;62
7.3.4.1;A. Yeasts (Saccharomyces cerevisiae and Schizosaccharomyces pombe);62
7.3.4.2;B. Nematodes (C. elegans);63
7.3.4.3;C. Drosophila Embryos;65
7.3.5;V. Postfreezing Processing;66
7.3.5.1;A. Freeze-Substitution;67
7.3.5.2;B. Embedding;70
7.3.5.3;C. FS and Embedding for Tomography;70
7.3.5.4;D. FS and Embedding for Immunocytochemistry;70
7.3.5.5;E. A.Case Study of Adjusting FS Variables to Improve Visualization;73
7.3.5.6;F. Sectioning;74
7.3.5.7;G. Microscopy and Evaluation of Results;75
7.3.5.8;H. Artifacts of HPF;77
7.3.6;VI. Summary;80
7.3.7;References;80
7.4;Chapter 3: Cryopreparation Methodology for Plant Cell Biology;85
7.4.1;I. Introduction;86
7.4.1.1;A. Historical Context;86
7.4.1.2;B. Techniques and Organisms Discussed Here;87
7.4.2;II. Rationale;87
7.4.2.1;A. Cryoimmobilization Versus Traditional Chemical Fixation at Ambient Temperatures: Considerations Based on First Principles;87
7.4.2.2;B. Why Plants and Fungi Are Usually Difficult to Fix, but Rather Easy to Freeze?;90
7.4.3;III. Methods;91
7.4.3.1;A. Cryoimmobilization;91
7.4.3.2;B. Freeze-Substitution.;93
7.4.3.3;C. Resin Embedding with Epoxides or LR White;94
7.4.3.4;D. Microtomy, Staining, and TEM;95
7.4.4;IV. Materials;96
7.4.5;V. Discussion;96
7.4.5.1;A. Cryoimmobilization: Choice of Freezing Method;96
7.4.5.2;B. Space Fillers for HPF;97
7.4.5.3;C. Freeze-Substitution;98
7.4.5.4;D. Embedding;110
7.4.5.5;E. FS and Embedding Media for Microanalysis and Autoradiography;114
7.4.5.6;F. Other Cryopreparation Techniques;117
7.4.6;VI. Concluding Remarks;118
7.4.7;Acknowledgments;119
7.4.8;References;119
7.5;Chapter 4: Correlative Light and Electron Microscopy of Early Caenorhabditis elegans Embryos in Mitosis;129
7.5.1;I. Introduction;130
7.5.2;II. Rationale;132
7.5.3;III. Methods;133
7.5.3.1;A. Staging of Isolated Embryos by LM;133
7.5.3.2;B. High-Pressure Freezing and Freeze-Substitution;135
7.5.3.3;C. Thin-Layer Embedding, Serial Sectioning, and Prescreening of Samples;136
7.5.3.4;D. Electron Tomography;138
7.5.4;IV. Instrumentation and Materials;139
7.5.4.1;A. Staging of Early Embryos;139
7.5.4.2;B. High-Pressure Freezing and Cryoprocessing of Staged Embryos;139
7.5.4.3;C. Thin-Layer Embedding, Serial Sectioning, and Prescreening of Samples;140
7.5.4.4;D. Electron Tomography;140
7.5.5;V. Discussion;140
7.5.5.1;A. Live-Cell Imaging;140
7.5.5.2;B. Capillary Tubes;141
7.5.5.3;C. Use of BSA as a "Filler";142
7.5.5.4;D. High-Pressure Freezing;142
7.5.5.5;E. Serial Sectioning;143
7.5.5.6;F. Electron Tomography and 3D Modeling;143
7.5.5.7;G. Concluding Remarks;145
7.5.6;Acknowledgments;145
7.5.7;References;145
8;Part II: Imaging Fixed Cells in Three Dimensions;149
8.1;Chapter 5: Understanding Microtubule Organizing Centers by Comparing Mutant and Wild-Type Structures with Electron Tomography;153
8.1.1;I. Introduction;154
8.1.2;II. Rationale;155
8.1.3;III. Methods;156
8.1.3.1;A. High-Pressure Freezing, Freeze-Substitution, and Embedding;156
8.1.3.2;B. Preparation of Sections for EM;158
8.1.3.3;C. Image Acquisition and Tomographic Reconstruction;158
8.1.3.4;D. 3D Modeling of the Basal Body Complex;163
8.1.4;IV. Instrumentation and Materials;166
8.1.4.1;A. High-Pressure Freezing and Freeze-Substitution;166
8.1.4.2;B. Electron Tomography;166
8.1.4.3;C. Tomographic Reconstruction and Modeling;167
8.1.5;V. Discussion;167
8.1.6;VI. Summary;169
8.1.7;Acknowledgments;169
8.1.8;References;169
8.2;Chapter 6: Whole-Cell Investigation of Microtubule Cytoskeleton Architecture by Electron Tomography;173
8.2.1;I. Introduction;174
8.2.2;II. Rationale;175
8.2.3;III. Methods;175
8.2.3.1;A. Sample Preparation;175
8.2.3.2;B. Tomogram Acquisition;179
8.2.3.3;C. Tomogram Calculation and 3D Model Reconstruction;183
8.2.3.4;D. Analysis of 3D Data;188
8.2.4;IV. Materials;189
8.2.4.1;A. Cells and Media;189
8.2.4.2;B. High-Pressure Freezer, Freeze-Substitution Resins;189
8.2.4.3;C. Microtome;190
8.2.4.4;D. Microscope Hardware;190
8.2.4.5;E. Computers;190
8.2.4.6;F. Software Package IMODcopy;190
8.2.5;V. Discussion;191
8.2.6;VI. Summary;193
8.2.7;Acknowledgments;193
8.2.8;References;193
8.3;Chapter 7: Electron Microscopy of Archaea;197
8.3.1;I. Introduction;198
8.3.2;II. Rationale;200
8.3.2.1;A. Selection of an Archaeon;200
8.3.2.2;B. Selection of Imaging Technology;200
8.3.2.3;C. Preparing Sulfolobus solfataricus for Electron Tomography;201
8.3.3;III. Methods and Materials;207
8.3.3.1;A. Cultivation of Sulfolobus solfataricus;207
8.3.3.2;B. Cryoprotectants;208
8.3.3.3;C. Concentrating Cells for High-Pressure Freezing;209
8.3.3.4;D. High-Pressure Freezing;209
8.3.3.5;E. Freeze Substitution, Embedding, and Sectioning;210
8.3.3.6;F. Fiducial Application;210
8.3.3.7;G. Imaging and Modeling Overview;210
8.3.3.8;H. Acquiring Tilt Series;212
8.3.3.9;I. Converting Tilt Series to Full-Cell Tomographic Reconstructions;213
8.3.3.10;J. Segmentation: Delineating Objects of Interest;213
8.3.4;IV. Discussion;217
8.3.5;V. Summary;217
8.3.6;References;218
8.4;Chapter 8: Reconstructing Mammalian Membrane Architecture by Large Area Cellular Tomography;221
8.4.1;I. Introduction and Rationale;222
8.4.2;II. Methods and Materials;224
8.4.2.1;A. Mammalian Cell and Tissue Culture;225
8.4.2.2;B. Fast-Freezing and Freeze-Substitution;228
8.4.2.3;C. Microtomy and Grid Preparation;229
8.4.2.4;D. Basic Instrumentation Requirements for ET of Thick Plastic Sections;229
8.4.2.5;E. Acquisition of Digital Tilt Series from Large Cellular Areas by Automated IVEM/HVEM Montaging Tomography;230
8.4.2.6;F. 3D Reconstruction of Large Cellular Areas;231
8.4.2.7;G. 3D Segmentation and Quantitative Analysis;236
8.4.3;III. Discussion;239
8.4.3.1;A. Future Directions;241
8.4.4;IV. Summary;243
8.4.5;Acknowledgments;243
8.4.6;References;244
8.5;Chapter 9: Visualization of Membrane-Cytoskeletal Interactions During Plant Cytokinesis;249
8.5.1;I. Introduction;250
8.5.2;II. Rationale;251
8.5.3;III. Materials and Methods;252
8.5.3.1;A. Processing Dividing Cells for ET;252
8.5.3.2;B. Visualizing Plant Cytokinesis by ET;254
8.5.3.3;C. Quantitative Analysis;262
8.5.4;IV. Discussion;265
8.5.5;V. Summary;266
8.5.6;References;267
8.6;Chapter 10: Electron Tomographic Methods for Studying the Chemical Synapse;269
8.6.1;I. Introduction and Rationale;270
8.6.2;II. Neurons, Neural Networks, and the Synapse;270
8.6.3;III. ET and the Synapse;271
8.6.4;IV. Types of Neurons and Synapses;272
8.6.4.1;A. Neuromuscular Junction Synapse;273
8.6.4.2;B. Ribbon Synapses;273
8.6.4.3;C. CNS Synapses;273
8.6.5;V. Sources of Neurons for Structural Studies;274
8.6.6;VI. Methods for Assessing Neuron and Synapse Integrity;275
8.6.7;VII. Rapid Freezing and HPF of Neurons;276
8.6.8;VIII. Modeling and Analysis of Synaptic Structures;277
8.6.8.1;A. Size and Organization of the Vesicle Pool;277
8.6.8.2;B. Organization of the Postsynaptic Density Proteins;279
8.6.8.3;C. Synaptic Cleft Complexes;279
8.6.8.4;D. Endosomal Compartments;279
8.6.8.5;E. Microtubule and Neurofilament Arrangements;279
8.6.9;IX. Stimulation-Dependent Changes in Synaptic Structure;280
8.6.10;X. The Future of EM Tomography and Synapses;280
8.6.11;XI. Conclusions;283
8.6.12;Acknowledgments;283
8.6.13;References;283
8.7;Chapter 11: Using Electron Microscopy to Understand Functional Mechanisms of Chromosome Alignment on the Mitotic Spindle;287
8.7.1;I. Introduction;288
8.7.1.1;A. Brief Review of Mitosis;289
8.7.1.2;B. Challenges in Studying the Mitotic Apparatus by Electron Microscopy;290
8.7.2;II. Basic Methodology;291
8.7.2.1;A. Conventional Fixation and Embedding;291
8.7.2.2;B. Cell Monolayers/Flat Embedding;292
8.7.2.3;C. High-Pressure Freezing and Freeze-Substitution;292
8.7.2.4;D. Electron Tomography;294
8.7.3;III. Organization of MTs in the Mitotic Spindle;295
8.7.3.1;A. Use of Serial Thin-Section Analysis to Determine the Spatial Organization of Spindle MTs;295
8.7.3.2;B. Use of Electron Tomography to Track MTs and Determine MT Dynamics;298
8.7.3.3;C. Determining the Polarity of Spindle MTs;299
8.7.4;IV. Deciphering Mechanisms of Chromosome Alignment and K-Fiber Formation Using Correlative Microscopy;300
8.7.4.1;A. Correlative LM/EM;300
8.7.4.2;B. Establishment of the Search and Capture Model for Chromosome Attachment;301
8.7.4.3;C. Elucidation of Mechanisms for Chromosome Congression;303
8.7.5;V. Toward a Molecular Understanding of Kinetochore Architecture;304
8.7.5.1;A. Kinetochore Function and Composition;304
8.7.5.2;B. The Classical Trilaminar Model of the Kinetochore Ultrastructure;306
8.7.5.3;C. Effects of Specimen Preparation on Kinetochore Ultrastructure;306
8.7.5.4;D. Immunolocalization of Kinetochore Components;306
8.7.5.5;E. Imaging of Isolated Protein Complexes from the Kinetochore;308
8.7.6;VI. Beyond Morphology: Use of Electron Tomography to Model Kinetochore Control of MT Dynamics;311
8.7.6.1;A. Nature of the Problem;311
8.7.6.2;B. Using High-Throughput Electron Tomography as an Assay for kMT Dynamics;311
8.7.6.3;C. A New Model for Kinetochore Control of MT Dynamics;314
8.7.6.4;D. Conventional Fixation Versus HPF/FS;314
8.7.7;VII. Conclusions and Future Directions;315
8.7.8;Acknowledgments;315
8.7.9;References;315
8.8;Chapter 12: Electron Microscopic Analysis of the Leading Edge in Migrating Cells;323
8.8.1;I. Introduction;324
8.8.2;II. Understanding the Mechanisms of Leading Edge Protrusion: Complementary Roles of LM and EM;325
8.8.3;III. Platinum Replica EM of the Leading Edge Cytoskeleton;329
8.8.3.1;A. Cell Culture;331
8.8.3.2;B. Extraction;334
8.8.3.3;C. Fixation;337
8.8.3.4;D. Dehydration and Critical Point Drying;339
8.8.3.5;E. Platinum/Carbon Coating;341
8.8.3.6;F. Release and Mounting of Replicas;342
8.8.4;IV. Identification of Cytoskeletal Components in Platinum Replica Preparations;343
8.8.4.1;A. Decoration of Actin Filaments with Myosin Subfragments;343
8.8.4.2;B. Immunostaining;344
8.8.5;Acknowledgments;344
8.8.6;References;344
8.9;Chapter 13: Imaging Actomyosin In Situ;349
8.9.1;I. Introduction and Overview;350
8.9.1.1;A. Why Electron Tomography?;350
8.9.1.2;B. Basic Features of Insect Flight Muscle;352
8.9.2;II. Preparing the Specimen;355
8.9.2.1;A. Materials;355
8.9.2.2;B. Mechanical Monitoring of Rapid Freezing;355
8.9.2.3;C. Fixation by Freeze-Substitution;358
8.9.2.4;D. Physical Limitations of Freezing;359
8.9.2.5;E. Validation of Specimen Preservation;361
8.9.3;III. Data Collection and Tomogram Calculation;362
8.9.3.1;A. Single- or Dual-Axis Tilt Series?;362
8.9.3.2;B. Tilt Angle Increment;363
8.9.3.3;C. Tilt-Series Alignment;364
8.9.3.4;D. Computing and Displaying the Tomogram;369
8.9.4;IV. Identifying Similar Structures in an Ensemble;370
8.9.4.1;A. Effect of the Missing Wedge;370
8.9.4.2;B. Selecting and Extracting Repeating Motifs;372
8.9.4.3;C. Alignment of Repeating Units;374
8.9.4.4;D. Reference-Based Alignment Schemes;375
8.9.4.5;E. Mask Construction;376
8.9.4.6;F. Correspondence Analysis of the Crossbridges;378
8.9.4.7;G. Number of Factors to Use;378
8.9.4.8;H. Number of Classes to Compute;378
8.9.4.9;I. Alternative Averaging Strategies;382
8.9.5;V. Building Atomic Models;383
8.9.6;VI. Relating Class Averages to the Specimen;386
8.9.7;VII. Application to IFM;388
8.9.8;VIII. Application to Other Types of Specimen;389
8.9.9;IX. Summary;390
8.9.10;Acknowledgments;390
8.9.11;References;391
9;Part III: Imaging Frozen-Hydrated Cells and Cell Parts;397
9.1;References;400
9.2;Chapter 14: Electron Tomography of Bacterial Chemotaxis Receptor Assemblies;401
9.2.1;I. Introduction;402
9.2.2;II. Analysis of Tsr Assemblies in Membrane Extracts;403
9.2.3;III. Electron Tomography of Fixed, Cryosectioned E. coli Cells;405
9.2.4;IV. Cryo-Electron Microscopy of Frozen-Hydrated Sections of E. coli Cells;406
9.2.5;V. Structure of Chemotaxis Receptors Using Cryo-Electron Tomography;409
9.2.6;VI. Summary;411
9.2.7;References;412
9.3;Chapter 15: How to "Read " a Vitreous Section;413
9.3.1;I. Introduction;414
9.3.2;II. Conventional Versus Cryo-Electron Microscopy: A Comparison;415
9.3.2.1;A. Vitreous Sections Are Unstained and Nonaggregated;416
9.3.2.2;B. Vitreous Section Are Globally Uniform;419
9.3.2.3;C. Vitreous Sections Are Locally Rich in Fine Structures;419
9.3.2.4;D. Cutting-Induced Deformation Is Inherent to Vitreous Sections;424
9.3.2.5;E. Vitreous Sections Are Beam Sensitive;427
9.3.3;III. Illustration: Two Pitfalls;428
9.3.3.1;A. Block Tectonics;428
9.3.3.2;B. Not Every Smooth Vitreous Section Is Admirable;431
9.3.4;Acknowledgments;431
9.3.5;References;431
9.4;Chapter 16: Single-Particle Electron Cryomicroscopy of the Ion Channels in the Excitation-Contraction Coupling ;435
9.4.1;I. Introduction;436
9.4.1.1;A. Excitation-Contraction Coupling in Striated Muscle;436
9.4.1.2;B. Cryo-EM of Single Particles;438
9.4.2;II. Single-Particle Cryo-EM Methodology;439
9.4.2.1;A. Specimen Preparation and Data Collection;439
9.4.2.2;B. CTF Correction;441
9.4.2.3;C. Image Data Quality Assessment;441
9.4.2.4;D. Image Processing and 3D Reconstruction;443
9.4.2.5;E. Current Limitations;445
9.4.2.6;F. Structural Feature Interpretation;445
9.4.2.7;G. Interpretation of Cryo-EM Maps by Fitting X-ray Structures of Components or Domains;446
9.4.3;III. 3D Reconstruction of Proteins in Triad Junction;446
9.4.3.1;A. Ryanodine Receptor;446
9.4.3.2;B. 3D Structure of DHPR;453
9.4.4;IV. Spatial Arrangement of DHPRs and RyRs in Triad Junctions;456
9.4.5;V. Summary;457
9.4.6;Acknowledgments;458
9.4.7;References;458
9.5;Chapter 17: Electron Microscopy of Microtubule-Based Cytoskeletal Machinery;465
9.5.1;I. Introduction;467
9.5.2;II. Rationale;470
9.5.3;III. Methods;471
9.5.3.1;A. Helical 3D Reconstruction;474
9.5.3.2;B. Back Projection;480
9.5.3.3;C. High-Resolution Metal Shadowing (HRMS);481
9.5.3.4;D. 3D Reconstructions from Single Particles;482
9.5.3.5;E. Cryo-ET of Microtubules Complexed with Kinesin or Axonemal Dynein;483
9.5.4;IV. Discussion;485
9.5.5;Acknowledgments;486
9.5.6;References;486
9.6;Chapter 18: Reconstructing the Endocytotic Machinery;491
9.6.1;I. Introduction;492
9.6.2;II. Rationale;493
9.6.3;III. Methods;495
9.6.3.1;A. Vitrification of Protein Assemblies;495
9.6.3.2;B. Single-Particle EM;496
9.6.3.3;C. Cryo-Electron Tomography;497
9.6.3.4;D. Docking Crystal Structures into EM Density Maps;498
9.6.4;IV. Results;498
9.6.4.1;A. The Human Tf-TfR Complex;498
9.6.4.2;B. Clathrin Coats;503
9.6.4.3;C. Clathrin-Coated Vesicles;507
9.6.5;V. Discussion;510
9.6.6;VI. Summary;512
9.6.7;References;513
10;Part IV: Localizing Macromolecules in Cells;517
10.1;References;519
10.2;Chapter 19: 3D Immunolocalization with Plastic Sections;521
10.2.1;I. Introduction;522
10.2.2;II. Rationale for the Various Approaches to Immuno-EM;523
10.2.3;III. Methods for Immuno-EM;524
10.2.3.1;A. Preembedding Methods;524
10.2.3.2;B. Postembedding Methods;525
10.2.3.3;C. Sample Preparation;525
10.2.4;IV. Comparison of the Methods;532
10.2.5;V. Summary;539
10.2.6;Acknowledgments;539
10.2.7;References;539
10.3;Chapter 20: Electron Microscopy Analysis of Viral Morphogenesis;543
10.3.1;I. Introduction;544
10.3.2;II. Analysis of Virus Assembly on Plastic Sections;545
10.3.2.1;A. Epon Embedding of Infected Cell Preparations: Technical Considerations;546
10.3.2.2;B. Assembly of the Human Cytomegalovirus;547
10.3.2.3;C. Assembly of SIV Virions and Virus-like Particles;549
10.3.3;III. Immunolabeling of Ultrathin Cryosections: Applications of the Tokuyasu Technique to Study Virus Assembly;552
10.3.3.1;A. Immunolabeling of Cryosections;553
10.3.3.2;B. An Example of Immunolabeling for EM: Assembly of SIV VLPs and Virions;555
10.3.3.3;C. Protocols for Double-Staining Immunolabeling;558
10.3.3.4;D. Double- and Triple-Labeling. Studies of HIV Assembly in Macrophages;561
10.3.3.5;E. Localization of Other Cellular and Viral Components;564
10.3.3.6;F. Quantification of Gold Particle Distributions;564
10.3.4;IV. Future Developments;565
10.3.5;V. Summary and Conclusions;566
10.3.6;Acknowledgments;567
10.3.7;References;567
10.4;Chapter 21: Electron Tomography of Immunolabeled Cryosections;571
10.4.1;I. Introduction;572
10.4.2;II. Methods;573
10.4.2.1;A. Specimen Fixation and Freezing;573
10.4.2.2;B. Cryoultramicrotomy;575
10.4.2.3;C. Immunolabeling of Cryosections;575
10.4.2.4;D. Immuno-Electron Microscopy and Tomography;576
10.4.3;III. Results and Discussion;577
10.4.4;References;585
10.5;Chapter 22: Visualizing Macromolecules with Fluoronanogold: From Photon Microscopy to Electron Tomography;587
10.5.1;I. Introduction;588
10.5.2;II. Experimental Approach for Immunostaining of pKi-67 and RNAP I;588
10.5.2.1;A. Cells;588
10.5.2.2;B. Fixation;589
10.5.2.3;C. Immunostaining;589
10.5.2.4;D. Confocal Microscopy;589
10.5.2.5;E. Electron Microscopy;589
10.5.3;III. Applications;590
10.5.3.1;A. pKi-67;591
10.5.3.2;B. RNAP I;597
10.5.4;IV. Discussion;599
10.5.5;Acknowledgments;601
10.5.6;References;601
10.6;Chapter 23: Markers for Correlated Light and Electron Microscopy;603
10.6.1;I. Introduction;604
10.6.2;II. How Do LM and EM Complement Each Other?;604
10.6.3;III. Fluorescence Photooxidation;605
10.6.3.1;A. Principles;605
10.6.3.2;B. Small, Organic Fluorophores;608
10.6.3.3;C. Eosin-Based Fluorescent Probes;609
10.6.3.4;D. Genetic-Based Tags;609
10.6.4;IV. Enzymatic-Based Methods;611
10.6.5;V. Particle-Based Methods for Protein Localization;612
10.6.5.1;A. Gold Particles;612
10.6.5.2;B. Quantum Dots;613
10.6.6;VI. Concluding Remarks;615
10.6.7;Acknowledgments;616
10.6.8;References;616
10.7;Chapter 24: Localizing Specific Elements Bound to Macromolecules by EFTEM;621
10.7.1;I. Introduction;622
10.7.2;II. General Principles;623
10.7.2.1;A. Electron Energy Loss Spectroscopy and Imaging;623
10.7.2.2;B. Quantitative Analysis and Correction for Thickness Effects;628
10.7.2.3;C. Detection Limits;630
10.7.2.4;D. Mapping Elemental Distributions in 3D;631
10.7.3;III. Applications;632
10.7.3.1;A. Visualizing Metals;632
10.7.3.2;B. Visualizing Nucleic Acid;637
10.7.4;IV. Summary and Future Directions;639
10.7.5;Acknowledgments;639
10.7.6;References;640
10.8;Chapter 25: Localization of Protein Complexes by Pattern Recognition;643
10.8.1;I. Introduction;644
10.8.2;II. Template Matching;644
10.8.2.1;A. Template Libraries for Visual Proteomics;644
10.8.2.2;B. Principles of Machine Learning and Pattern Recognition;646
10.8.2.3;C. Templates;647
10.8.2.4;D. The Cross-Correlation. Function;648
10.8.2.5;E. Normalization of the Correlation Function;651
10.8.2.6;F. Local Correlation Functions;653
10.8.2.7;G. Fourier-Space Representation;655
10.8.2.8;H. Probabilistic Interpretation;656
10.8.2.9;I. Practical Implementation;657
10.8.3;III. The Missing-Wedge Problem;659
10.8.3.1;A. Tomographic Imaging;659
10.8.3.2;B. Restricted Cross-Correlation Coefficients;660
10.8.4;IV. Applications;661
10.8.5;V. Conclusions;663
10.8.6;References;665
11;Part V: Aspects of Data Collection and Analysis;667
11.1;References;670
11.2;Chapter 26: The Application of Energy-Filtered Electron Microscopy to Tomography of Thick, Selectively Stained Biological Samples;671
11.2.1;I. Introduction;672
11.2.2;II. Introduction to MPL Imaging;674
11.2.2.1;A. Motivation;674
11.2.2.2;B. Description of the Method;675
11.2.3;III. Detailed Description of MPL Tomography;676
11.2.4;IV. Techniques;679
11.2.5;V. Results;681
11.2.6;VI. Conclusions;685
11.2.7;Acknowledgments;686
11.2.8;References;686
11.3;Chapter 27: Optimization of Image Collection for Cellular Electron Microscopy;689
11.3.1;I. Introduction: Assessing the Role of Image Collection in Cellular Electron Microscopy;690
11.3.1.1;A. The Electron Microscope Image Collection Chain and the Role of the Detector;690
11.3.1.2;B. Quantitative Assessment of Imaging Performance Using Spectral Signal-to-Noise Ratio;691
11.3.2;II. Applications: The Importance and Meaning of SSNR in Cell Biology Applications;693
11.3.2.1;A. Tomography;693
11.3.2.2;B. Localization of Functional Units Within the Cell;698
11.3.3;III. Optimization of Detectors and of the Detection Process for Maximization of SSNR;703
11.3.3.1;A. Background: The Transition from Film to Electronic Detectors and the Value of Gain Normalization;703
11.3.3.2;B. The Physical Causes of SSNR Performance Reduction in Detectors;708
11.3.3.3;C. Specimen-Free Detector Characterization: DQE;711
11.3.3.4;D. Performance Trade-Off Example: Scintillator Thickness;717
11.3.3.5;E. Performance Trade-Off Example: Microscope Magnification and Sampling Choices;721
11.3.4;IV. Detectors: Proven Technologies;726
11.3.4.1;A. Scintillator Fiber-Optically Coupled to a CCD;727
11.3.4.2;B. Scintillator with Low-Sensitivity Lens Coupling to a CCD;728
11.3.5;V. Detectors: Experimental Technologies;729
11.3.5.1;A. High-Sensitivity Substrate-Less Transmission Scintillator Camera;730
11.3.5.2;B. Related Technologies for Contrast Enhancement: Zero-Loss Filtering, Higher Microscope Accelerating Voltage, and the Boersch Phase Plate;734
11.3.5.3;C. Direct Detection;739
11.3.5.4;D. Electron Image Deceleration;740
11.3.6;VI. Discussion;742
11.3.7;VII. Conclusions;743
11.3.8;Acknowledgments;743
11.3.9;References;744
11.4;Chapter 28: Future Directions for Camera Systems in Electron Microscopy;749
11.4.1;I. Introduction;750
11.4.2;II. Background and Rationale;755
11.4.2.1;A. Results of PAD Tests;755
11.4.3;III. Description of the Direct Detection Detector;757
11.4.4;IV. Detector Characterization;758
11.4.4.1;A. Noisy Pixels;758
11.4.4.2;B. Signal to Noise;759
11.4.4.3;C. Spatial Resolution;760
11.4.5;V. Radiation Damage;762
11.4.6;VI. Discussion;764
11.4.7;Acknowledgments;765
11.4.8;References;765
11.5;Chapter 29: Structure Determination In Situ by Averaging of Tomograms;769
11.5.1;I. Introduction;770
11.5.2;II. Tomography and Its Increase in Resolution by Averaging;773
11.5.2.1;A. The Fundamentals of Tomography;773
11.5.2.2;B. Resolution of Cryoelectron Tomograms;774
11.5.2.3;C. Increasing the Resolution of Tomograms by Averaging;775
11.5.3;III. Coherent Averaging of Macromolecules in Practice;778
11.5.3.1;A. Acquisition of Cryoelectron Tomograms;778
11.5.3.2;B. An Algorithm for Averaging Membrane-Bound Assemblies;779
11.5.4;IV. Applications of Tomogram Averaging;785
11.5.4.1;A. Purified Macromolecules;785
11.5.4.2;B. Highly Symmetric Assemblies;787
11.5.4.3;C. Assembly Structures Determined In Situ;787
11.5.5;V. Conclusion and Outlook;788
11.5.6;Acknowledgments;791
11.5.7;References;791
11.6;Chapter 30: Methods for Image Segmentation in Cellular Tomography;797
11.6.1;I. Introduction;798
11.6.2;II. Problem Formulation;799
11.6.3;III. Challenges in Cellular Tomography Segmentation;801
11.6.3.1;A. Data Set Variety;801
11.6.3.2;B. Low and Nonuniform Contrast;801
11.6.3.3;C. Interfering Structures;801
11.6.3.4;D. Low Signal-to-Noise Ratio;802
11.6.3.5;E. Anisotropic Resolution;803
11.6.3.6;F. Specimen Preparation Artifacts;804
11.6.4;IV. Important Considerations for Segmentation Methods;804
11.6.4.1;A. Automation and Efficiency;805
11.6.4.2;B. Input Parameters;805
11.6.4.3;C. Computer Requirements;806
11.6.4.4;D. Objectivity;806
11.6.5;V. How Do We Know if the Segmentation Method Is Reliable?;806
11.6.5.1;A. Measuring True/False Positives and Negatives;807
11.6.5.2;B. Invariance;808
11.6.5.3;C. Consistency;808
11.6.6;VI. Segmentation Methods for Cellular Tomography;809
11.6.6.1;A. 2D Versus 3D Methods;809
11.6.6.2;B. Variational Methods;809
11.6.6.3;C. The Watershed Transform;812
11.6.6.4;D. Wavelets and Brushlets;813
11.6.6.5;E. Nonlinear Diffusion Methods;813
11.6.6.6;F. Geometric Diffusions;817
11.6.6.7;G. Hysteresis Thresholding;817
11.6.7;VII. Orientation-Based Segmentation;818
11.6.7.1;A. The Importance of Scale and Orientations;818
11.6.7.2;B. Orientation Estimation;819
11.6.7.3;C. Orientation Filtering;820
11.6.8;VIII. Results Using Orientation-Based Segmentation;821
11.6.8.1;A. Method;822
11.6.8.2;B. Results;822
11.6.9;IX. Summary;825
11.6.10;Acknowledgments;825
11.6.11;References;825
11.7;Chapter 31: Database Resources for Cellular Electron Microscopy;827
11.7.1;I. Introduction;828
11.7.1.1;A. What Is a Database?;828
11.7.1.2;B. Why Are Images Different?;830
11.7.1.3;C. Web-Accessible Databases for EM;833
11.7.1.4;D. Data Management for EM;833
11.7.2;II. The Cell Centered Database Project;835
11.7.2.1;A. CCDB Search and Display;838
11.7.2.2;B. Multiscale Spatial Registration of CCDB Data;840
11.7.2.3;C. Database Interoperability;842
11.7.2.4;D. Data Input into the CCDB;845
11.7.3;III. Looking Ahead;847
11.7.4;Acknowledgments;848
11.7.5;References;848
12;Index;851
13;Volumes in Series;871