Advanced Techniques in Biological Electron Microscopy II: Specific Ultrastructural Probes

The use of the term "advanced" in the title of this book is somewhat ar­ bitrary and very much relative with respect to time. Many techniques which were considered at the "cutting edge" of ultrastructural methodology just a few years ago are now rou tin ely used in numerous laboratories. One could cite freeze-fracture, cryothin sectioning, or indeed most of the field of scan­ ning electron microscopy as concrete examples. Thus the use of the term "ad­ vanced techniques" must be interpreted with regard to the present state of the art, and is useful only in informing the potential reader that this volume is not a primer to be used as an initial introduction into basic biological elec­ tron microscopy. Many excellent volumes have filled that niche in the past few years, and it is not intended that this modest book be a complete com­ pendium of the field. Furthermore, any limited selection of papers on advanc­ ed techniques necessarily reflects the preferences and arbitrary whims of the editor, thereby excluding many equally important procedures which the knowledgeable reader will readily identify. The first volume of this series appeared approximately five years ago and illustrated techniques which were thought to represent advanced and yet ba­ sically morphological methods for gaining increased ultrastructural informa­ tion from biological specimens. The present volume, on the other hand, stresses techniques which provide specific physicochemical data on the speci­ mens in addition to the structural information.

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.