Methods of Preparation for Electron Microscopy: An Introduction for the Biomedical Sciences

Methods of Preparation for Electron Microscopy: An Introduction for the Biomedical Sciences

Paperback(Softcover reprint of the original 1st ed. 1987)

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Product Details

ISBN-13: 9783540175926
Publisher: Springer Berlin Heidelberg
Publication date: 07/27/1987
Edition description: Softcover reprint of the original 1st ed. 1987
Pages: 190
Product dimensions: 6.69(w) x 9.61(h) x 0.02(d)

Table of Contents

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

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