Histological Techniques for Electron Microscopy

Preface to the First EditionPreface to the Second Edition1. Organization and Management of an Electron Microscope Laboratory 1.1. Laboratory Layout 1.2. Equipment 1.3. Management of the Laboratory 1.4. Service Personnel 1.5. Technical Help l.6. Shared Facilities 1.7. Educating Administrators and Clinicians2. Tissue Exposure 2.1. Introductory Remarks 2.2. Post-Mortem Change 2.3. Minced Tissue 2.4. Topical, In Situ, Preservation of Natural Surfaces 2.5. In Situ Fixation of Cut Surfaces 2.6. Vascular Perfusion 2.7. Hyaluronidase Pretreatment 2.8. Special Problems with Human Tissues 2.9. Special Problems with Tissue Cultures and Cell Suspensions 2.10. The Use of Cold-Blooded and Young Animals 2.11. Botanical Material Literature Cited3 . Fixation 3.1. Introductory Remarks 3.2. Palade (1952) Fixative, Buffered Osmium Tetroxide 3.3. Variations of Osmium Tetroxide Fixatives 3.4. Special Purpose Osmium Tetroxide Fixatives 3.5. Temperature Control of Osmium Tetroxide Fixation 3.6. Duration of Osmium Tetroxide Fixation 3.7. Criteria of Good Osmium Tetroxide Fixation 3.8. Aldehyde Fixation: Introductory Remarks 3.9. Buffered Aldehydes as Primary Fixatives 3.10. Aldehydes as Fixatives for Histochemistry 3.11. Morphological Features of Aldehyde Fixation 3.12. The Permanganate Fixative of Luft (1956) 3.13. Rapid Freezing 3.14. Freeze-Drying 3.15. Freeze-Substitution Literature Cited4. Embedding 4.1. Introductory Remarks 4.2. Dehydration 4.3. Transitional Solvents 4.4. Infiltration 4.5. Handling Thick Embedding Media 4.6. Desirable Characteristics of the Embedment 4.7. Methacrylate Embedding 4.8. "Polymerization Damage" and the Use of "Prepolymerized" Methacrylate 4.9. Limitations of Methacrylate 4.10. Araldite Embedding 4.11. Epon Embedding 4.12. Maraglas Embedding 4.13. Polyester Vestopale Embedding 4.14. Selectron, Rigolac and Viapale Embedding 4.15. Cross-linked Methacrylate Embedding 4.16. Water-soluble Resin Embedding 4.17. Mixed Plastic Embedding 4.18. Gelatin Embedding 4.19. Preparing the Block for Sectioning Literature Cited5. Sectioning 5.1. Introductory Remarks 5.2. The Original Porter-Blum Microtome 5.3. Modification of the Porter-Blum Microtome 5.4. LKB "Ultrotome" 5.5. Leitz Ultramicrotome 5.6. Reichert Ultramicrotome 5.7. Cambridge Ultramicrotome 5.8. SI-RO-FLEX Ultramicrotome 5.9. JUM-5 Ultramicrotome 5.10. Sorvall MT-2 Ultramicrotome 5.11. Clevenger's (1963) Microtome 5.12. Knives Used for Ultrathin Sectionin 5.13. Glass Knife Manufacture 5.14. Knife Angle 5.15. Examination and Selection of Glass Knives 5.16. Diamond Knives 5.17. Trough Construction 5.18. Mounting the Knife 5.19. Preferred Orientation of Block in Microtome 5.20. Section Flotation 5.21. Flattening Sections 5.22. Sectioning 5.23. Contamination—Principal Cause of Sectioning Difficulty! 5.24. Estimation of Section Thickness 5.25. Summary of Sectioning Technique 5.26. Picking Up Sections Literature Cited6. Section Mounting 6.1. Introductory Remarks 6.2. Parlodion Films 6.3. Formvar Films 6.4. Preparing Film Nets 6.5. The Mechanics of Carbon Evaporation 6.6. Carbon Stabilization of Naked Sections 6.7. Carbon Stabilization of Plastic Films 6.8. Pure Carbon Films Prepared on a Plastic Substrate 6.9. Stripping Pure Carbon Films 6.10. "Sandwiched" Sections 6.11. Serial Sections 6.12. Mounting Screens in the Microscope Specimen Holder 6.13. Dirty Specimens Literature Cited7. "Staining" 7.1. Introductory Remarks 7.2. Penetration of Stains 7.3. Alkaline Lead Hydroxide Stains 7.4. Uranyl Stains 7.5. Phosphotungstic Acid Stain 7.6. Lawn's Permanganate Stain 7.7. Vanadium Salt Stains 7.8. Chromyl Chloride Stain 7.9. Stain Dependence upon Fixation 7.10. Histochemical Applications 7.11. Specific Antibody "Stains" 7.12. Autoradiography 7.13. Thick Sections for Conventional Microscopy 7.14. Removal of Cured Epoxy Resins Literature Cited8. Microscopy 8.1. Introductory Remarks 8.2. Objective Aperture Position and Size 8.3. Objective Aperture Alignment 8.4. Aperture Cleaning and Manufacture 8.5. Microscope Maintenance 8.6. Alignment, General Remarks 8.7. Objective Alignment 8.8. Alignment of the Illuminating System 8.9. Source Alignment 8.10. Condenser Alignment 8.11. Filament Replacement and Alignment 8.12. Objective Lens Compensation 8.13. Instrument Stability 8.14. Trouble Shooting General References9. Photography 9.1. Introductory Remark 9.2. Section Thickness versus Magnification 9.3. Focus 9.4. Special Considerations in Picture Taking 9.5. Reasons for Unsatisfactory Pictures 9.6. Photographic Plates 9.7. Plate Development 9.8. Enlarging 9.9. Timing Print Exposures 9.10. "Dodging" 9.11. Problem Negatives, Reduction and Intensificaion 9.12. Interpretation of Electron Microscope Images General Reference10. Particulate Specimens , Mounting, Shadowing, and Replication 10.1. Introductory Remarks 10.2. Suspended and Fragmented Material 10.3. Shadow-Casting 10.4. Replication 10.5. Single-Stage Replication 10.6. Preshadowed Replicas 10.7. Two-Stage Replication 10.8. Preparation of Surfaces for Replication 10.9. Materials for Replication, Deposited from Solution or Melt 10.10. Materials for Replication, Deposited by Vacuum Evaporation Literature Cited General References11. Particulate Specimens, Negative Staining 11.1. Introductory Remarks 11.2. Theoretical Aspects of Negative Staining 11.3. Negative Stains 11.4. Specimen Preparation for Negative Staining 11.5. Microscopy of Negatively Stained Material Literature CitedAppendix A. Outline of Basic Technique Training ProgramAppendix B. The Literature of Electron Microscopy Bibliographies Published Records of International Meetings Published Records of Major National MeetingsAppendix C. Some Sources of Equipment and Materials General Sources Specimen Grids Apertures Calibration SuppliesAuthor IndexSubject Index