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).- Bright Field Electron Microscopy.- Low Dose Transmission Electron Microscopy.- Dark Field Electron Microscopy.- 1.1.2 Conventional Scanning Electron Microscopy (SEM).- Imaging with Secondary and Back-Scattered Electrons.- 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.- General Comments.- Fixatives: Properties and Preparation.- Composition of Fixation Solutions.- The Fixation of Animal Cells.- The Fixation of Plants and Microorganisms.- The Fixation of Isolated Organelles.- Fixing for Immunocytochemistry.- 2.1.2 Dehydration.- 2.1.3 Embedding.- Embedding Media: General Usage and Precautions.- Conventional Embedding.- Water-Soluble Embedding Media.- Embedding for Immunocytochemistry.- Embedding Moulds and Specimen Orientation.- Embedding of Monolayer Cell Cultures.- 2.2 Ultramicrotomy.- 2.2.1 Trimming of Blocks.- General.- Controlled Trimming: Production and Staining of Semi-Thin Sections.- 2.2.2 Preparing Glass Knives.- Preparation of the Glass Strips.- Breaking Glass Squares.- Making Knives.- Judging the Quality of a Glass Knife.- Attaching Troughs.- Storing Glass Knives.- 2.2.3 Diamond Knives and Their Care.- 2.2.4 Conventional Sectioning.- Trough Liquids.- Using an Ultramicrotome.- Section Thickness.- Picking Up Sections.- Sectioning Problems.- 2.2.5 Cryo-ultramicrotomy.- Freezing the Sample.- Sectioning the Frozen Sample.- Picking up Frozen Sections.- 2.2.6 Staining Sections.- Staining Solutions.- Procedure for Double Staining Sections.- Staining Sections of Material Embedded for Immunocytochemical Purposes.- Staining Cryosections.- Block Staining.- 2.3 Macromolecular EM.- 2.3.1 Isolated Proteins and Protein Aggregates.- Preparation of Specimens.- Negative Staining Techniques.- High Resolution Metal Shadowing.- Preparation and Imaging of Two-Dimensional Protein Crystals.- Making a “Tilt Series”.- 2.3.2 Isolated Nucleic Acids.- Problems and Aims.- Specimen Preparation.- Spreading and Diffusion Techniques Which Employ Cytochrome c.- “BAC” Technique.- Partial Denaturating, Heteroduplex and R-Loop Techniques.- 2.3.3 Nucleic Acid-Protein Complexes.- Specimen Preparation.- Production and Staining of NA-Protein Complexes.- 2.4 Immunoelectron Microscopy (I EM).- 2.4.1 Principle Requirements.- Antigens.- Antibodies.- 2.4.2 Labelling of Antigens in Cells and Cell Fractions.- Ferritin-Labelled Antibodies.- Immunolabelling with Protein A-Gold.- 2.4.3 Localization of Protein Subunits with Specific IgG Antibodies.- Preparation and Visualization of the Protein-Antibody Complex.- 2.5 Autoradiography.- 2.5.1 General Background.- Physical Basis.- Chemical Basis.- 2.5.2 Choice and Dosis of Radioactive Compounds.- Choosing a Radioactive Precursor.- 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.- Apparatus Required.- Choice of Emulsion; Consequences for Resolution.- Preparation of Sections.- LM Autoradiography.- Emulsion, Coating Techniques.- 2.5.6 Exposing, Developing and Fixing.- Exposing.- Developing and Fixing.- Future Developments in Autoradiography.- 2.6 Freeze (Fracturing) Etching.- 2.6.1 Introduction.- 2.6.2 Freezing.- Theoretical Background.- Cyroprotectants.- Supports.- Cryogens and Freezing Methods.- Storage of Frozen Specimens.- 2.6.3 Fracturing.- Transfer of the Object into the Vacuum Recipient.- The Fracturing Process.- Fracture Planes in Biological Material.- 2.6.4 Etching.- The Purpose of Etching.- Theory and Practice.- 2.6.5 Shadowing and Replica Formation.- Resistance-Heating Evaporation.- Electron Beam Evaporation.- 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.- Cleaning Surfaces.- 3.1.3 Stabilization.- Chemical Fixations.- Cryofixation.- 3.1.4 Dehydration.- 3.1.5 Drying.- Critical Point Drying.- Freeze Drying.- 3.1.6 Mounting Specimens.- 3.1.7 Increasing Conductivity.- Sputtering.- 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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