Elsevier S & T

Biomedical Electron Microscopy: Illustrated Methods and Interpretations

Maunsbach, Arvid B.; Afzelius, Björn A.

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Table of contents
  • Cover
  • Contentsv
  • FOREWORDxi
  • PREFACExiii
  • ACKNOWLEDGMENTSxv
  • CHAPTER 1. MICROGRAPH INTERPRETATION1
  • 1. Classical Preparation Method2
  • 2. Low Temperature Approach4
  • 3. A Common Test Specimen6
  • 4. Detection of Objects8
  • 5. Identification of Artifacts10
  • 6. Analysis of Geometry12
  • 7. Biological Identification14
  • 8. Biological Diversity16
  • 9. Analysis of Dynamics: Endocytosis18
  • 10. Analysis of Dynamics: Synthesis20
  • 11. Comparison of Methods22
  • 12. Variations in Magnifications24
  • 13. Interpretation Difficulties26
  • 14. Diagnostic Pathology28
  • CHAPTER 2. FIXATIVES31
  • 1. Osmium Tetroxide and Glutaraldehyde at Low Magnification32
  • 2. Osmium Tetroxide and Glutaraldehyde at High Magnification34
  • 3. Glutaraldehyde Concentration: Perfusion Fixation36
  • 4. Glutaraldehyde Concentration: Immersion Fixation38
  • 5. Long Fixation Times40
  • 6. Formaldehyde–Glutaraldehyde Combinations42
  • 7. Potassium Permanganate, Picric Acid, and Ruthenium Red44
  • 8. Lead Salts and Tannic Acid46
  • 9. Uranyl Acetate Postfixation48
  • 10. Tannic Acid–Uranyl Acetate Variations50
  • 11. Osmium Tetroxide–Potassium Ferrocyanide52
  • 12. Osmium Tetroxide Artifacts54
  • 13. Glutaraldehyde Artifacts56
  • CHAPTER 3. FIXATIVE VEHICLE59
  • 1. Absence and Presence of Buffer60
  • 2. Comparison of Buffers62
  • 3. Osmolality of Perfusion Fixatives64
  • 4. Effects of Osmolality on Cell Shape66
  • 5. Effects of Osmolality on Cell Organelles68
  • 6. Adjustment of Osmolality with Sucrose70
  • 7. Colloid Osmotic Pressure: Low Magnification72
  • 8. Colloid Osmotic Pressure: High Magnification74
  • 9. Phosphate Buffer Precipitate76
  • CHAPTER 4. FIXATIVE APPLICATION79
  • 1. Perfusion–Fixation versus Immersion–Fixation80
  • 2. Perfusion–Fixation with Pressure Control82
  • 3. Fixation by Dripping in Vivo84
  • 4. Immersion–Fixation86
  • 5. Variability within the Tissue88
  • 6. Unsuccessful Perfusion–Fixation90
  • 7. Superficial Tissue Damage92
  • 8. Early Postmortal Changes94
  • 9. Late Postmortal Changes96
  • 10. Influence of Biopsy Method98
  • 11. Microwave Treatment100
  • CHAPTER 5. DEHYDRATION AND EMBEDDING103
  • 1. Stepwise versus Direct Dehydration104
  • 2. Prolonged Dehydration in Ethanol106
  • 3. Prolonged Dehydration in Acetone108
  • 4. Inert Dehydration110
  • 5. Choice of Intermediate Solvent112
  • 6. Epon, Araldite, and Vestopal: Unstained Sections114
  • 7. Epon, Araldite, and Vestopal: Stained Sections116
  • 8. Different Brands of Epoxy Resins118
  • 9. Spurr and LR White120
  • 10. Embedding of Isolated Cells122
  • CHAPTER 6. FREEZING AND LOW-TEMPERATURE EMBEDDING125
  • 1. Plunge Freezing126
  • 2. Contact Freezing of Unfixed Tissue128
  • 3. Contact Freezing of Fixed Tissue130
  • 4. High-Pressure Freezing132
  • 5. Freeze-Substitution in Methanol/Uranyl Acetate134
  • 6. Freeze-Substitution in Osmium Tetroxide Acetone136
  • 7. Progressive Lowering of Temperature Embedding in Lowicryl138
  • CHAPTER 7. SUPPORT FILMS141
  • 1. Surface Topography142
  • 2. Stability of Film or Section144
  • 3. Holey Films146
  • 4. Thick and Thin Support Films148
  • 5. Folds in Support Film150
  • 6. Defects in Formvar Films152
  • 7. Common Contaminants154
  • 8. Volatile Contamination156
  • CHAPTER 8. ULTRAMICROTOMY159
  • 1. Correlation of Light and Electron Microscopy160
  • 2. Section Thickness: Low Magnification162
  • 3. Section Thickness: High Magnification164
  • 4. Section Thickness: Half-Micron Section166
  • 5. Determination of Section Thickness168
  • 6. Folds in the Section170
  • 7. Collection of Sections172
  • 8. Surface Topography of Sections174
  • 9. Knife Scratches176
  • 10. Mottling and Flaking178
  • 11. Worn Glass Knives180
  • 12. Transmitted Vibrations182
  • 13. Vibrations and Knife Marks184
  • 14. Selective Chatter186
  • 15. Compression188
  • 16. Holes and Deformations190
  • 17. Contamination during Microtomy192
  • 18. Extraction during Sectioning194
  • 19. Cryoultramicrotomy: Survey Sections196
  • 20. Collection of Cryosections198
  • 21. Thickness of Cryosections200
  • 22. Staining of Cryosections202
  • 23. Defects in Cryosections204
  • CHAPTER 9. SECTION-STAINING207
  • 1. Lead Citrate Staining208
  • 2. Uranyl Acetate Staining210
  • 3. Enhanced Section Staining212
  • 4. Effects of Grid Storage214
  • 5. Section Exposed to Electron Beam216
  • 6. Effect of Electron Beam218
  • 7. Lead-Staining Granularity220
  • 8. Contamination222
  • 9. Block-Staining Precipitate224
  • 10. Removal of Contamination226
  • CHAPTER 10. MICROSCOPY229
  • 1. Resolving Power230
  • 2. Through-Focus Series: Hole and Latex Particle232
  • 3. Through-Focus Series: Myelin Sheath234
  • 4. Through-Focus Series: Cells236
  • 5. Minimum Contrast Focusing238
  • 6. Wobbler Focusing240
  • 7. Accelerating Voltages 20–100 kV242
  • 8. Accelerating Voltages 80–200 kV244
  • 9. Unsaturated Electron Beam246
  • 10. Condenser Apertures248
  • 11. Objective Aperture250
  • 12. Through-Focus Series: Astigmatism252
  • 13. Image Distortion254
  • 14. Chromatic Aberration256
  • 15. Mechanical Instability258
  • 16. Specimen Drift versus Astigmatism260
  • 17. Focus Drift262
  • 18. Electrical Instabilities264
  • 19. Contamination in the Electron Beam266
  • 20. Radiation Damage268
  • 21. Radiation Damage and Contamination270
  • 22. Low-Dose Exposure272
  • 23. Spectroscopic Imaging: Thin Film274
  • 24. Spectroscopic Imaging: Thick Section276
  • 25. Spectroscopic Imaging: Carbon278
  • 26. Spectroscopic Imaging: Calcium280
  • 27. Spectroscopic Imaging: Contrast Changes282
  • 28. Cryoelectron Microscopy: Na,K-ATPase Crystals284
  • 29. Defects in Cryoelectron Micrographs286
  • CHAPTER 11. IMAGE RECORDING289
  • 1. Exposure Time290
  • 2. Over/Underexposure292
  • 3. Effects of Development294
  • 4. Exposure Dose Adjustment296
  • 5. Primary Magnification298
  • 6. Damage to Negatives300
  • 7. Damage to Wet Negatives302
  • 8. Film/Imaging Plate/Charge-Coupled Device (CCD) Camera304
  • 9. Enlarged Digital Recordings306
  • 10. Variation in Electron Dose308
  • 11. Corrections of CCD Camera310
  • CHAPTER 12. PHOTOGRAPHIC AND DIGITAL PRINTING313
  • 1. Photographic Paper of Different Grades314
  • 2. Multigrade Paper316
  • 3. Exposure and Development318
  • 4. Enlargement of Micrograph Details320
  • 5. Objective Lens in Enlarger322
  • 6. Focusing of Enlarger324
  • 7. Intermediate Diapositive326
  • 8. Errors in Photographic Printing328
  • 9. Retouch330
  • 10. Comparison of Printers: Low Magnification332
  • 12. Pixel Size at Printing336
  • CHAPTER 13. NEGATIVE STAINING339
  • 1. Negative Staining Methods340
  • 2. Properties of Support Film342
  • 3. Comparison of Stains344
  • 4. Thickness of Stain346
  • 5. Concentration of Specimen348
  • 6. Deformation of Specimen350
  • 7. Radiation Damage352
  • CHAPTER 14. AUTORADIOGRAPHY355
  • 1. Undeveloped Emulsion356
  • 2. Developed Emulsion358
  • 3. Resolution360
  • 4. Quantitation362
  • 5. Preparatory Defects364
  • CHAPTER 15. CYTOCHEMISTRY367
  • 1. Influence of Fixation368
  • 2. Preincubation Treatment370
  • 3. Appearance of Reaction Product372
  • 4. Composition of Incubation Medium374
  • 5. Cytochemical Resolution376
  • 6. Unspecific Staining378
  • 7. Extraction of Reaction Product380
  • CHAPTER 16. IMMUNOCYTOCHEMISTRY383
  • 1. Fixation of Sensitive Antigens384
  • 2. Fixation of Insensitive Antigens386
  • 3. Comparison of Embedding Media388
  • 4. Influence of Preincubation Solutions390
  • 5. Comparison of Primary Antibodies392
  • 6. Dilution of Primary Antibody394
  • 7. Quantitation of Gold Particles396
  • 8. Controls398
  • 9. Comparison of Gold Probes400
  • 10. Amplification of Gold Particles402
  • 11. Section Staining404
  • 12. Resolution406
  • 13. Background Labeling408
  • 14. Antigen Retrieval by Etching410
  • 15. Antigen Retrieval with Sodium Dodecyl Sulfate412
  • 16. Double Labeling414
  • 17. Immunonegative Staining416
  • 18. Freeze-Fracture Replica Labeling418
  • 19. Preembedding Labeling420
  • 20. Semithin Light Microscopic Sections422
  • CHAPTER 17. FREEZE FRACTURING AND SHADOWING427
  • 1. Shadowing of DNA Molecules428
  • 2. Shadowing of Protein Molecules430
  • 3. Freeze-Fractured Membrane Faces432
  • 4. Thickness of Replica: Low Magnification434
  • 5. Thickness of Replica: High Magnification436
  • 6. Rotary Shadowing438
  • 7. Complementary Replicas and Stereo Images440
  • 8. Ice Crystals and Etching442
  • 9. Quick-Freeze Deep Etching444
  • 10. Identification of Transport Molecules446
  • 11. Contamination448
  • 12. Plastic Distortion450
  • 13. Replica Defects452
  • CHAPTER 18. SAMPLING AND QUANTITATION455
  • 1. Calibration of Magnification456
  • 2. Sampling and Object Variability458
  • 3. Sampling of Pellets: Differential Centrifugation460
  • 4. Sampling of Pellets: Gradient Centrifugation462
  • 5. Micrograph Montages464
  • 6. Automated Digital Montages466
  • 7. Resolution of Digital Montages468
  • 8. Measurements on Digital Images470
  • 9. Stereological Grids472
  • 10. Cycloid Test System474
  • CHAPTER 19. IMAGE PROCESSING477
  • 1. Digital Contrast Changes478
  • 2. Processing of Scanned Image480
  • 3. Translational Image Enforcement482
  • 4. Averaging of Macromolecular Assemblies484
  • 5. Rotational Image Enforcement486
  • 6. Photographic versus Computer Averaging488
  • 7. Fourier Correction of Section Chatter490
  • 8. Removal of Image Defects492
  • 9. Scientific Fraud: Removal of Objects494
  • 10. Scientific Fraud: Manipulation of Labeling496
  • CHAPTER 20. THREE-DIMENSIONAL RECONSTRUCTIONS499
  • 1. Comparison between Transmission and Scanning Electron Microscopy500
  • 2. Serial Sectioning502
  • 3. Large Three-Dimensional Objects504
  • 4. Tilting of Section Cell Nucleus506
  • 5. Tilting of Section" Nuclear Envelope508
  • 6. Helical Structures510
  • 7. Computer-Analyzed Helices512
  • 8. A Three-Dimensional Model of Na, K-ATPase514
  • APPENDIX: PRACTICAL METHODS517
  • 1. Fixation517
  • 2. Dehydration and Embedding522
  • 3. Low Temperature Embedding524
  • 4. Support Films526
  • 5. Ultramicrotomy528
  • 6. Section Staining530
  • 7. Microscopy and Image Recording532
  • 8. Photographic Work533
  • 9. Negative Staining534
  • 10. Autoradiography535
  • 11. Immunolabeling536
  • 12. Freeze Fracture538
  • AUTHOR BIOGRAPHIES539
  • ACKNOWLEDGMENTS FOR REPRODUCTION OF FIGURES541
  • INDEX545
Book details
  • Vendor Elsevier S & T
  • SKU 9780124806108
  • ISBN-13 9780080528090
  • Author Maunsbach, Arvid B.; Afzelius, Björn A.
  • Category Science
  • Subject Electron Microscopes & Microscopy

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Description eBook delivery & access Reviews
This comprehensive reference illustrates optimal preparation methods in biological electron microscopy compared with common methodological problems. Not only will the basic methodologies of transmission electron microscopy like fixation, microtomy, and microscopy be presented, but the authors also endeavor to illustrate more specialized techniques such as negative staining, autoradiography, cytochemistry, immunoelectron microscopy, and computer-assisted image analysis.

Key Features
* Authored by the key leaders in the biological electron microscopy field
* Illustrates both optimal and suboptimal or artifactual results in a variety of electron microscopy disciplines
* Introduces students on how to read and interpret electron micrographs