Biomedical Electron Microscopy (eBook)

Front Cover 1
BIOMEDICAL ELECTRON MICROSCOPY 4
Copright Page 5
FOREWORD 12
PREFACE 14
ACKNOWLEDGMENTS 16
CHAPTER 1. MICROGRAPH INTERPRETATION 18
1. Classical Preparation Method 19
2. Low Temperature Approach 21
3. A Common Test Specimen 23
4. Detection of Objects 25
5. Identification of Artifacts 27
6. Analysis of Geometry 29
7. Biological Identification 31
8. Biological Diversity 33
9. Analysis of Dynamics: Endocytosis 35
10. Analysis of Dynamics: Synthesis 37
11. Comparison of Methods 39
12. Variations in Magnifications 41
13. Interpretation Difficulties 43
14. Diagnostic Pathology 45
CHAPTER 2. FIXATIVES 48
1. Osmium Tetroxide and Glutaraldehyde at Low Magnification 49
2. Osmium Tetroxide and Glutaraldehyde at High Magnification 51
3. Glutaraldehyde Concentration: Perfusion Fixation 53
4. Glutaraldehyde Concentration: Immersion Fixation 55
5. Long Fixation Times 57
6. Formaldehyde–Glutaraldehyde Combinations 59
7. Potassium Permanganate, Picric Acid, and Ruthenium Red 61
8. Lead Salts and Tannic Acid 63
9. Uranyl Acetate Postfixation 65
10. Tannic Acid–Uranyl Acetate Variations 67
11. Osmium Tetroxide–Potassium Ferrocyanide 69
12. Osmium Tetroxide Artifacts 71
13. Glutaraldehyde Artifacts 73
CHAPTER 3. FIXATIVE VEHICLE 76
1. Absence and Presence of Buffer 77
2. Comparison of Buffers 79
3. Osmolality of Perfusion Fixatives 81
4. Effects of Osmolality on Cell Shape 83
5. Effects of Osmolality on Cell Organelles 85
6. Adjustment of Osmolality with Sucrose 87
7. Colloid Osmotic Pressure: Low Magnification 89
8. Colloid Osmotic Pressure: High Magnification 91
9. Phosphate Buffer Precipitate 93
CHAPTER 4. FIXATIVE APPLICATION 96
1. Perfusion–Fixation versus Immersion–Fixation 97
2. Perfusion–Fixation with Pressure Control 99
3. Fixation by Dripping in Vivo 101
4. Immersion–Fixation 103
5. Variability within the Tissue 105
6. Unsuccessful Perfusion–Fixation 107
7. Superficial Tissue Damage 109
8. Early Postmortal Changes 111
9. Late Postmortal Changes 113
10. Influence of Biopsy Method 115
11. Microwave Treatment 117
CHAPTER 5. DEHYDRATION AND EMBEDDING 120
1. Stepwise versus Direct Dehydration 121
2. Prolonged Dehydration in Ethanol 123
3. Prolonged Dehydration in Acetone 125
4. Inert Dehydration 127
5. Choice of Intermediate Solvent 129
6. Epon, Araldite, and Vestopal: Unstained Sections 131
7. Epon, Araldite, and Vestopal: Stained Sections 133
8. Different Brands of Epoxy Resins 135
9. Spurr and LR White 137
10. Embedding of Isolated Cells 139
CHAPTER 6. FREEZING AND LOW-TEMPERATURE EMBEDDING 142
1. Plunge Freezing 143
2. Contact Freezing of Unfixed Tissue 145
3. Contact Freezing of Fixed Tissue 147
4. High-Pressure Freezing 149
5. Freeze-Substitution in Methanol/Uranyl Acetate 151
6. Freeze-Substitution in Osmium Tetroxide Acetone 153
7. Progressive Lowering of Temperature Embedding in Lowicryl 155
CHAPTER 7. SUPPORT FILMS 158
1. Surface Topography 159
2. Stability of Film or Section 161
3. Holey Films 163
4. Thick and Thin Support Films 165
5. Folds in Support Film 167
6. Defects in Formvar Films 169
7. Common Contaminants 171
8. Volatile Contamination 173
CHAPTER 8. ULTRAMICROTOMY 176
1. Correlation of Light and Electron Microscopy 177
2. Section Thickness: Low Magnification 179
3. Section Thickness: High Magnification 181
4. Section Thickness: Half-Micron Section 183
5. Determination of Section Thickness 185
6. Folds in the Section 187
7. Collection of Sections 189
8. Surface Topography of Sections 191
9. Knife Scratches 193
10. Mottling and Flaking 195
11. Worn Glass Knives 197
12. Transmitted Vibrations 199
13. Vibrations and Knife Marks 201
14. Selective Chatter 203
15. Compression 205
16. Holes and Deformations 207
17. Contamination during Microtomy 209
18. Extraction during Sectioning 211
19. Cryoultramicrotomy: Survey Sections 213
20. Collection of Cryosections 215
21. Thickness of Cryosections 217
22. Staining of Cryosections 219
23. Defects in Cryosections 221
CHAPTER 9. SECTION-STAINING 224
1. Lead Citrate Staining 225
2. Uranyl Acetate Staining 227
3. Enhanced Section Staining 229
4. Effects of Grid Storage 231
5. Section Exposed to Electron Beam 233
6. Effect of Electron Beam 235
7. Lead-Staining Granularity 237
8. Contamination 239
9. Block-Staining Precipitate 241
10. Removal of Contamination 243
CHAPTER 10. MICROSCOPY 246
1. Resolving Power 247
2. Through-Focus Series: Hole and Latex Particle 249
3. Through-Focus Series: Myelin Sheath 251
4. Through-Focus Series: Cells 253
5. Minimum Contrast Focusing 255
6. Wobbler Focusing 257
7. Accelerating Voltages 20–100 kV 259
8. Accelerating Voltages 80–200 kV 261
9. Unsaturated Electron Beam 263
10. Condenser Apertures 265
11. Objective Aperture 267
12. Through-Focus Series: Astigmatism 269
13. Image Distortion 271
14. Chromatic Aberration 273
15. Mechanical Instability 275
16. Specimen Drift versus Astigmatism 277
17. Focus Drift 279
18. Electrical Instabilities 281
19. Contamination in the Electron Beam 283
20. Radiation Damage 285
21. Radiation Damage and Contamination 287
22. Low-Dose Exposure 289
23. Spectroscopic Imaging: Thin Film 291
24. Spectroscopic Imaging: Thick Section 293
25. Spectroscopic Imaging: Carbon 295
26. Spectroscopic Imaging: Calcium 297
27. Spectroscopic Imaging: Contrast Changes 299
28. Cryoelectron Microscopy: Na,K-ATPase Crystals 301
29. Defects in Cryoelectron Micrographs 303
CHAPTER 11. IMAGE RECORDING 306
1. Exposure Time 307
2. Over/Underexposure 309
3. Effects of Development 311
4. Exposure Dose Adjustment 313
5. Primary Magnification 315
6. Damage to Negatives 317
7. Damage to Wet Negatives 319
8. Film/Imaging Plate/Charge-Coupled Device (CCD) Camera 321
9. Enlarged Digital Recordings 323
10. Variation in Electron Dose 325
11. Corrections of CCD Camera 327
CHAPTER 12. PHOTOGRAPHIC AND DIGITAL PRINTING 330
1. Photographic Paper of Different Grades 331
2. Multigrade Paper 333
3. Exposure and Development 335
4. Enlargement of Micrograph Details 337
5. Objective Lens in Enlarger 339
6. Focusing of Enlarger 341
7. Intermediate Diapositive 343
8. Errors in Photographic Printing 345
9. Retouch 347
10. Comparison of Printers: Low Magnification 349
12. Pixel Size at Printing 353
CHAPTER 13. NEGATIVE STAINING 356
1. Negative Staining Methods 357
2. Properties of Support Film 359
3. Comparison of Stains 361
4. Thickness of Stain 363
5. Concentration of Specimen 365
6. Deformation of Specimen 367
7. Radiation Damage 369
CHAPTER 14. AUTORADIOGRAPHY 372
1. Undeveloped Emulsion 373
2. Developed Emulsion 375
3. Resolution 377
4. Quantitation 379
5. Preparatory Defects 381
CHAPTER 15. CYTOCHEMISTRY 384
1. Influence of Fixation 385
2. Preincubation Treatment 387
3. Appearance of Reaction Product 389
4. Composition of Incubation Medium 391
5. Cytochemical Resolution 393
6. Unspecific Staining 395
7. Extraction of Reaction Product 397
CHAPTER 16. IMMUNOCYTOCHEMISTRY 400
1. Fixation of Sensitive Antigens 401
2. Fixation of Insensitive Antigens 403
3. Comparison of Embedding Media 405
4. Influence of Preincubation Solutions 407
5. Comparison of Primary Antibodies 409
6. Dilution of Primary Antibody 411
7. Quantitation of Gold Particles 413
8. Controls 415
9. Comparison of Gold Probes 417
10. Amplification of Gold Particles 419
11. Section Staining 421
12. Resolution 423
13. Background Labeling 425
14. Antigen Retrieval by Etching 427
15. Antigen Retrieval with Sodium Dodecyl Sulfate 429
16. Double Labeling 431
17. Immunonegative Staining 433
18. Freeze-Fracture Replica Labeling 435
19. Preembedding Labeling 437
20. Semithin Light Microscopic Sections 439
CHAPTER 17. FREEZE FRACTURING AND SHADOWING 444
1. Shadowing of DNA Molecules 445
2. Shadowing of Protein Molecules 447
3. Freeze-Fractured Membrane Faces 449
4. Thickness of Replica: Low Magnification 451
5. Thickness of Replica: High Magnification 453
6. Rotary Shadowing 455
7. Complementary Replicas and Stereo Images 457
8. Ice Crystals and Etching 459
9. Quick-Freeze Deep Etching 461
10. Identification of Transport Molecules 463
11. Contamination 465
12. Plastic Distortion 467
13. Replica Defects 469
CHAPTER 18. SAMPLING AND QUANTITATION 472
1. Calibration of Magnification 473
2. Sampling and Object Variability 475
3. Sampling of Pellets: Differential Centrifugation 477
4. Sampling of Pellets: Gradient Centrifugation 479
5. Micrograph Montages 481
6. Automated Digital Montages 483
7. Resolution of Digital Montages 485
8. Measurements on Digital Images 487
9. Stereological Grids 489
10. Cycloid Test System 491
CHAPTER 19. IMAGE PROCESSING 494
1. Digital Contrast Changes 495
2. Processing of Scanned Image 497
3. Translational Image Enforcement 499
4. Averaging of Macromolecular Assemblies 501
5. Rotational Image Enforcement 503
6. Photographic versus Computer Averaging 505
7. Fourier Correction of Section Chatter 507
8. Removal of Image Defects 509
9. Scientific Fraud: Removal of Objects 511
10. Scientific Fraud: Manipulation of Labeling 513
CHAPTER 20. THREE-DIMENSIONAL RECONSTRUCTIONS 516
1. Comparison between Transmission and Scanning Electron Microscopy 517
2. Serial Sectioning 519
3. Large Three-Dimensional Objects 521
4. Tilting of Section Cell Nucleus 523
5. Tilting of Section" Nuclear Envelope 525
6. Helical Structures 527
7. Computer-Analyzed Helices 529
8. A Three-Dimensional Model of Na, K-ATPase 531
APPENDIX: PRACTICAL METHODS 534
1. Fixation 534
2. Dehydration and Embedding 539
3. Low Temperature Embedding 541
4. Support Films 543
5. Ultramicrotomy 545
6. Section Staining 547
7. Microscopy and Image Recording 549
8. Photographic Work 550
9. Negative Staining 551
10. Autoradiography 552
11. Immunolabeling 553
12. Freeze Fracture 555
AUTHOR BIOGRAPHIES 556
ACKNOWLEDGMENTS FOR REPRODUCTION OF FIGURES 558
INDEX 562