Methods of preparation for electron microscopy : an introduction for the biomedical sciences

摘要: 1 An Introduction to Electron Microscopy (EM).- 1.1 Imaging Methods in Microscopy.- 1.1.1 Conventional Transmission (TEM).- 1.1.1.1 Bright Field 1.1.1.2 Low Dose 1.1.1.3 Dark 1.1.2 Scanning (SEM).- 1.1.2.1 with Secondary and Back-Scattered Electrons.- 1.1.2.2 at Accelerating Voltages.- 1.2 Preparation Procedures TEM.- 1.2.1 Overview.- 1.2.2 Structural Preservation During Fixation, Dehydration Embedding of Biological Objects.- 1.3 Problems.- 1.3.1 On the Interpretation TEM Images.- 1.3.2 SEM 1.4 Support Films.- 1.4.1 Grids for Their Pretreatment.- 1.4.2 Formvar 1.4.3 Collodion 1.4.4 Hydrophilisation 1.4.5 Films Holes.- 1.4.6 Carbon 2 2.1 Embedding.- 2.1.1 Chemical Fixations.- 2.1.1.1 General Comments.- 2.1.1.2 Fixatives: Properties Preparation.- 2.1.1.3 Composition Fixation Solutions.- 2.1.1.4 The Animal Cells.- 2.1.1.5 Plants Microorganisms.- 2.1.1.6 Isolated Organelles.- 2.1.1.7 Fixing Immunocytochemistry.- 2.1.2 Dehydration.- 2.1.3 2.1.3.1 Media: Usage Precautions.- 2.1.3.2 2.1.3.3 Water-Soluble Media.- 2.1.3.4 2.1.3.5 Moulds Specimen Orientation.- 2.1.3.6 Monolayer Cell Cultures.- 2.2 Ultramicrotomy.- 2.2.1 Trimming Blocks.- 2.2.1.1 General.- 2.2.1.2 Controlled Trimming: Production Staining Semi-Thin Sections.- 2.2.2 Preparing Glass Knives.- 2.2.2.1 Strips.- 2.2.2.2 Breaking Squares.- 2.2.2.3 Making 2.2.2.4 Judging Quality a Knife.- 2.2.2.5 Attaching Troughs.- 2.2.2.6 Storing 2.2.3 Diamond Knives Care.- 2.2.4 Sectioning.- 2.2.4.1 Trough Liquids.- 2.2.4.2 Using an Ultramicrotome.- 2.2.4.3 Section Thickness.- 2.2.4.4 Picking Up 2.2.4.5 Sectioning 2.2.5 Cryo-ultramicrotomy.- 2.2.5.1 Freezing Sample.- 2.2.5.2 Frozen 2.2.5.3 up 2.2.6 2.2.6.1 2.2.6.2 Procedure Double 2.2.6.3 Sections Material Embedded Immunocytochemical Purposes.- 2.2.6.4 Cryosections.- 2.2.6.5 Block Staining.- 2.3 Macromolecular EM.- 2.3.1 Proteins Protein Aggregates.- 2.3.1.1 Specimens.- 2.3.1.2 Negative Techniques.- 2.3.1.3 High Resolution Metal Shadowing.- 2.3.1.4 Two-Dimensional Crystals.- 2.3.1.5 "Tilt Series".- 2.3.2 Nucleic Acids.- 2.3.2.1 Problems Aims.- 2.3.2.2 2.3.2.3 Spreading Diffusion Techniques Which Employ Cytochrome c.- 2.3.2.4 "BAC" Technique.- 2.3.2.5 Partial Denaturating, Heteroduplex R-Loop 2.3.3 Acid-Protein Complexes.- 2.3.3.1 2.3.3.2 NA-Protein 2.4 Immunoelectron (I EM).- 2.4.1 Principle Requirements.- 2.4.1.1 Antigens.- 2.4.1.2 Antibodies.- 2.4.2 Labelling Antigens Cells Fractions.- 2.4.2.1 Ferritin-Labelled 2.4.2.2 Immunolabelling A-Gold.- 2.4.3 Localization Subunits Specific IgG 2.4.3.1 Visualization Protein-Antibody Complex.- 2.5 Autoradiography.- 2.5.1 Background.- 2.5.1.1 Physical Basis.- 2.5.1.2 2.5.2 Choice Dosis Radioactive Compounds.- 2.5.2.1 Choosing Precursor.- 2.5.2.2 Dosage.- 2.5.3 Working Isotopes-Radiation Protection.- 2.5.4 Radio-Labelled Cells/Tissues 2.5.5 Photographic Emulsions 2.5.5.1 Apparatus Required.- 2.5.5.2 Emulsion Consequences Resolution.- 2.5.5.3 2.5.5.4 LM 2.5.5.5 Emulsion, Coating 2.5.6 Exposing, Developing Fixing.- 2.5.6.1 Exposing.- 2.5.6.2 2.5.6.3 Future Developments 2.6 Freeze (Fracturing) Etching.- 2.6.1 Introduction.- 2.6.2 Freezing.- 2.6.2.1 Theoretical 2.6.2.2 Cyroprotectants.- 2.6.2.3 Supports.- 2.6.2.4 Cryogens Methods.- 2.6.2.5 Storage 2.6.3 Fracturing.- 2.6.3.1 Transfer Object into Vacuum Recipient.- 2.6.3.2 Fracturing Process.- 2.6.3.3 Fracture Planes Material.- 2.6.4 2.6.4.1 Purpose 2.6.4.2 Theory Practice.- 2.6.5 Shadowing Replica Formation.- 2.6.5.1 Resistance-Heating Evaporation.- 2.6.5.2 Beam 2.6.5.3 Measurement 2.6.6 Cleaning Replica.- 2.6.7 Artifacts 2.6.8 Freeze-Etch Machine: Practical Description.- 3 SEM.- 3.1 3.1.1 3.1.2 Size Handling Specimens Exposing Surfaces.- 3.1.2.1 3.1.3 Stabilization.- 3.1.3.1 3.1.3.2 Cryofixation.- 3.1.4 3.1.5 Drying.- 3.1.5.1 Critical Point 3.1.5.2 3.1.6 Mounting 3.1.7 Increasing Conductivity.- 3.1.7.1 Sputtering.- 3.1.7.2 Evaporating.- 3.2 3.3 Demonstration Surfaces via Replicas Casts.- 3.4 Internal Through Dry-Fracturing (Dry-Cleaving).- 3.5 Element Analysis.- 4 Evaluation Micrographs.- 4.1 Morphometry.- 4.1.1 4.1.2 Measurement: Some Points.- 4.1.3 Stereology: Principles.- 4.1.4 Collection Data Statistical Treatments.- 4.2 Averaging Image Reconstruction.- 4.2.1 4.2.2 Markham Rotation.- 4.2.3 Principles Light Optical Diffraction.- 4.2.4 Computer-Assisted Appendix: Buffers Microscopy.