Advanced Techniques in Biological Electron Microscopy II

Ultrastructural Localization of Lectin Receptors.- A. Introduction.- B. Purification of Lectins.- C. Purification of Markers.- I. Hemocyanin.- II. Ferritin.- 1. Cadmium Sulfate Crystallization.- 2. Ammonium Sulfate Precipitation.- 3. Ultracentrifugation.- III. Peroxidase.- IV. Mannan-Iron Complex.- D. Synthesis of Probes and Labeling Techniques.- I. Lectin-Hemocyanin.- 1. Labeling Procedures.- 2. Platinum-Carbon Replicas.- II. Lectin-Ferritin Conjugates.- 1. One-Step Glutaraldehyde Coupling.- 2. Two-Step Glutaraldehyde Method.- 3. Labeling Procedures.- III. Lectin-Peroxidase Techniques.- 1. Two-Step Lectin-Peroxidase Labeling.- 2. Single-Step Lectin-Peroxidase Labeling.- IV. Lectin-Polysaccharide-Iron Complexes.- 1. Lectin-Dextran-Iron Complexes.- 2. Lectin-Mannan-Iron Complexes.- References.- Antibody-labeling Techniques.- A. Rationale.- B. Antibody Labels.- C. Methods for Coupling Label to Antibody.- I. One-Step Method.- II. Two-Step Method.- D. Iodination of Antibody.- I. Iodination of Antibody with Chloramine T.- II. 125I-Labeled Antibody for Transmission Electron Microscopy.- III. Lactoperoxidase Labeling of Antibody.- IV. Antibody Labeling with an Acylating Agent.- E. Hemocyanin Label of Antibody.- I. Purification of Hemocyanin.- II. Conjugation of Hemocyanin with Antibody.- F. Reaction of Antibody with Cells.- G. Clotting Procedure for Handling Single Cell Suspensions.- H. Radioautography.- I. Replica Techniques.- I. Surface Replica Technique.- II. Freeze-etching Technique.- J. Conclusions.- References.- Cell Surface Labeling for the Scanning Electron Microscope.- A. Introduction.- B. Labeling Techniques for the SEM.- I. The Label.- II. The Marker.- 1. Electron-Dense Markers.- 2. Markers Recognizable by Their Shapes.- 3. Cathodoluminescent and Other Markers.- III. Coupling Label to Marker.- 1. Direct Coupling.- 2. Indirect Coupling.- 3. Purification and Analysis of Conjugates.- C. Interpretation of Cell Surface Labeling in the SEM.- I. Quantitation.- 1. Influence of Valence of the Label.- 2. Stoichiometry of the Binding of Label to Marker.- 3. Influence of the Size of the Marker.- II. Resolution.- 1. Size of the Marker.- 2. Size of the Label-Marker Complex.- III. The Sample.- 1. Label-Induced Rearrangements.- 2. Sources of ‘False’ Labeling.- 3. Types of Samples.- 4. Subsequent Sample Preparation for the SEM.- D. Summary.- References.- Low-Temperature Biological Scanning Electron Microscopy.- A. Introduction.- B. Low-Temperature Solidification of Cell and Tissue Fluids.- C. Pre-treatment Before the Cooling Process.- I. Chemical Fixation.- II. Artificial Nucleators.- III. Cryoprotection.- IV. Embedding Agents.- V. Non-chemical Pre-treatment.- D. Specimen Cooling.- E. Post-freezing Preparative Procedures.- I. Frozen-dried or Frozen-hydrated.- II. External Surfaces of Internal Details.- F. Specimen Transfer.- G. Low-temperature Specimen Stages.- H. Specimen Examination.- I. Conclusions.- References.- Quantitative Electron Microscopy of Nucleic Acids.- A. Introduction.- B. Basic Protein Film Method.- I. Aqueous Technique.- II. Formamide Technique.- III. Reagents.- IV. Problems Related to Contrast.- V. Double-Strand/Single-Strand Distinction and Length Ratios.- C. Heteroduplex Molecules.- I. Experimental Procedure.- II. Examples.- III. Complications Which May Arise in Constructing and Examining Heteroduplex Molecules.- IV. Branch Migration.- V. Terminology, Topology, and Stability of Branch Points.- VI. Diheteroduplexes.- VII. Partial Sequence Homology.- VIII. Partial Denaturation Mapping.- D. Measuring and Error Analysis.- I. Measurement Procedures.- II. Reference Markers and Orientation.- III. Error Analysis.- IV. Determination of Number Average Molecular Weight..- 1. DNA Standard.- 2. Unbiased Sampling of Molecules.- 3. Background Subtraction of Contaminating DNA Molecules.- V. Determination of DNA Concentration by Electron Microscopy.- E. Artifacts and Topology Considerations.- I. Flowers.- II. Lateral Aggregation.- III. Intermolecular Overlap.- IV. Branch Peelback in Heteroduplex Molecules.- V. 2:2 Branch Point Configuration.- VI. Renaturation of Single-Stranded Circular Molecules.- VII. Topologic Restriction to Renaturation in Linear Molecules — Renaturation of ‘Knotted’ DNA.- F. RNA and Transcription Complexes.- I. Techniques for Preparing RNA.- II. Secondary Structure Maps.- III. Transcription Complexes.- IV. Mapping of Complementary RNA Sequences in DNA.- 1. R-Loop Method.- 1. Single-Strand Binding Protein Method.- G. Tagging Methods.- I. RNA-Ferritin Tags.- II. Protein-Ferritin Tags.- F. General Comments and Problems.- H. Protein-free Spreading.- I. Direct Visualization.- II. Intercalating Dye Method.- III. Benzyldimethylalkylammonium Chloride Method.- IV. Other Methods.- References.- Electron Microscopy of Specific Proteins: Three-Dimensional Mapping of Ribosomal Proteins Using Antibody Labels.- A. Introduction.- B. Techniques.- C Interpretation.- References.- Electron Microscopy and Electron Diffraction Studies on Hydrated Membranes.- A. Introduction.- B. Operation of Hydration Chamber in an Electron Microscope.- I. Hydration Chamber.- 1. Principles.- 2. Chambers for Fixed-beam Transmission Electron Microscope.- 3. Chambers for Scanning Electron Microscope.- II. Preparation of Wet Membrane Specimens.- C. Electron Microscopy.- I. Dark Field.- II. Energy Filters.- III. Image Intensifies.- D. Electron Diffraction (ED).- I. Selective Area Electron Diffraction.- II. Small-angle Electron Diffraction.- III. Phase Transition and Phase Separation in Membranes.- E. Conclusions and Future Development.- References.