Advanced Techniques in Biological Electron Microscopy 3

Three-Dimensional Reconstruction of Nonperiodic Macromolecular Assemblies from Electron Micrographs.- 1 Introduction.- 1.1 General.- 1.2 The Three-Dimensional Structure as an Average.- 2 Methods for Obtaining Projection Data.- 2.1 What is Being Reconstructed?.- 2.2 Scanning of Projection Data.- 2.3 Hardware and Software for Electron Image Processing.- 2.4 Alignment of Projections.- 2.5 Methods for Obtaining Statistically Significant Projections.- 3 Methods of Three-Dimensional Reconstruction.- 3.1 Preliminaries.- 3.2 Some Existing Algorithms for Reconstruction.- 3.2.1 Real-Space Methods.- 3.2.2 Fourier Methods.- 3.3 Resolution of the Reconstructed Object.- 3.4 The Point Spread Function and the Effect of Angular Limitations.- 3.5 Conical Tilting Geometry.- 3.6 Experimental Data Collection Methods.- 3.6.1 Goniometer Tilting.- 3.6.2 Use of Multiple A Priori Orientations.- 3.6.3 Random Tilts of a Long, Quasi-Cylindrical Particle.- 3.6.4 Random In-Plane Rotations of a Particle Having a Fixed Orientation.- 3.7 Representation of Three-Dimensional Data.- 4 Experimental Results.- 4.1 Ribosome.- 4.2 Oxygen-Carrying Proteins: Hemocyanins and Hemoglobins of Invertebrates.- 4.3 Chromatin.- 4.4 Survey of Published Results.- 5 Conclusions.- Appendix: Some Important Definitions and Theorems.- A.1 The Two-Dimensional Fourier Transform.- A.2 Convolution.- A.3 Resolution in the Fourier Domain.- A.4 Low-Pass Filtration.- A.5 Correlation Functions.- A.6 The Projection Theorem.- References.- High Resolution Biological X-Ray Microanalysis of Diffusable Ions.- 1 Introduction.- 2 Principles of the Technique.- 3 Specimen Preparation.- 3.1 Introduction.- 3.2 Low Temperature Methods for AEM.- 3.3 Tissue Preparation.- 3.4 Rapid Freezing.- 3.5 Cryoultramicrotomy.- 3.6 Frozen Transfer.- 3.7 Freezing Drying.- 4 Quantitation.- 4.1 Theory of Quantitation of Biological Thin Samples.- 4.2 Errors in Quantitation.- 4.3 Mapping.- 5 Applications in the Analysis of Diffusable Ions.- 6 Conclusion.- References.- Metal Deposition by High-Energy Sputtering for High Magnification Electron Microscopy.- 1 Introduction.- 1.1 Increase of Surface Information.- 1.2 Background.- 1.2.1 Enhancement of Topographic Contrast.- 1.2.2 Increase of Contrast at High Magnification.- 1.2.3 High Resolution Replicas.- 1.2.4 Scanning Electron Microscopy.- 1.2.5 Visualization of Surface Fine Structures.- 1.3 Contrast Principles.- 1.4 Limiting Properties of Conventionally Used Metal Films.- 1.4.1 Metal Film Thickness.- 1.4.2 Particle Decoration.- 2 Methods.- 2.1 Microscopy.- 2.1.1 Specimens and Preparation.- 2.1.2 Instruments.- 2.1.3 Useful Magnifications.- 2.2 Metal Deposition Technique.- 2.2.1 High Vacuum Metal Deposition System.- 2.2.2 Geometric Factors for Metal Deposition.- 2.2.3 Film Thickness Measurements.- 3 Metal Deposition.- 3.1 Metal Film Formation.- 3.1.1 Phases of Film Growth.- 3.1.2 Nucleation.- 3.1.3 Film Growth from Nuclei.- 3.2 Low-Rate High-Energy Sputter Deposition.- 3.2.1 Energy Parameters.- 3.2.2 Low-Rate Deposition.- 3.3 Thin Continuous Film Production.- 3.3.1 Reduction of Decoration Effects.- 3.3.2 Reduction of Scattering.- 3.3.3 Reduction of Self-Shadowing.- 3.3.4 Reduction of Contamination.- 3.3.5 Critical Film Thickness.- 3.4 Coating Strategy.- 3.4.1 Surface Information.- 3.4.2 Rationales for Continuous Film Application.- 3.4.3 Choice of Coating Technique.- 3.4.4 Test Specimen.- 4 Conclusion.- References.- Computer Programs for Biological Stereology.- 1 Introduction.- 1.1 Stereology: An Overview.- 1.2 Biological Applications of Stereology.- 2 Point Counting Programs.- 2.1 Point Counting Stereology: An Overview.- 2.2 Hardware.- 2.3 Software.- 2.3.1 Experiment Design.- 2.3.2 Data Input.- 2.3.3 Data Management.- 2.3.4 Data Analysis.- 2.3.5 Data Output.- 3 Digitizing Programs.- 3.1 Digitizing Stereology: An Overview.- 3.2 Hardware.- 3.3 Software.- 3.3.1 Data Collected.- 3.3.2 Data Analysis.- 4 Special Purpose Stereology Programs.- 4.1 Size Frequency Distributions and Mean Caliper Diameters.- 4.1.1 Convex Structures.- 4.1.2 Nonconvex Structures.- 4.1.3 Oriented Structures.- 4.2 Sampling Analysis.- 4.3 Quantification of Freeze-Fracture Replicas.- 4.4 Pattern Analysis.- 5 Concluding Comments.- References.- A Guide to Fracture Label: Cytochemical Labeling of Freeze-Fractured Cells.- 1 Introduction.- 1.1 Freeze-Fracture: Membrane Splitting.- 1.2 Freeze Etching: Membrane Cytochemistry.- 1.3 Fracture Label: Freeze-Fracture Cytochemistry.- 2 Experimental Procedures.- 2.1 Main Steps.- 2.2 Preparation of Specimens.- 2.2.1 Fixation.- 2.2.2 Embedding in BSA.- 2.2.3 Impregnation.- 2.2.4 Freezing.- 2.2.5 The Sandwich Method.- 2.3 Freeze Fracture.- 2.3.1 Thin-Section Fracture Label (TS-FL).- 2.3.2 Critical Point Drying Fracture Label (CPD-FL).- 2.4 Thawing and Deglycerination.- 2.4.1 Thin-Section Fracture Label (TS-FL).- 2.4.2 Critical Point Drying Fracture Label (CPD-FL).- 2.5 Labeling.- 2.5.1 Detection of Concanavalin A Binding Sites.- 2.5.2 Detection of Wheat-Germ Agglutinin Binding Sites.- 2.6 Processing of Labeled Specimes for Electron Microscopy.- 2.6.1 Thin-Section Fracture Label.- 2.6.2 Critical Point Drying Fracture Label.- 3 Electron Microscopy.- 3.1 Thin-Section Fracture Label.- 3.2 Critical Point Drying Fracture Label.- 4 Interpretation.- 4.1 Postfracture Reorganization of Membrane Components.- 4.2 Labeling of Outer Surface Receptors on Protoplasmic Membrane Halves.- 4.3 Labeling of Intracellular Membranes and Nuclear Matrix.- 4.4 Commentary.- References.- The Preparation of Colloidal Gold Probes and Their Use as Marker in Electron Microscopy.- 1 Introduction.- 2 The Preparation and Storage of Colloidal Gold Sols.- 2.1 Introductory Remarks.- 2.2 Citrate Gold (15 nm). (Reducing Agent = Sodium Citrate).- 2.2.1 Stock Solutions.- 2.2.2 Procedure.- 2.2.3 Variation of the Size by Using Different Citrate Concentrations.- 2.3 A Modified Citrate Method for Producing 8-10 nm Gold.- 2.4 Phosphorous Gold (Reducing Agent = White Phosphorous).- 2.4.1 Introductory Remarks.- 2.4.2 Stock Solutions.- 2.4.3 Procedure.- 2.5 The Alternative Citrate-Tannic Acid Procedure for Preparing Small Gold Particles.- 2.5.1 The Procedure of Mulpordt.- 2.5.2 The Procedure of Slot and Geuze.- 3 The Production, Purification, and Storage of Gold Probes.- 3.1 General Remarks About the Adsorption of Proteins to Colloidal Gold.- 3.2 Preparation of Protein Solution and Colloidal Gold Sol Before the Adsorption Step.- 3.3 Determination of the Minimal Protecting Amount of Protein.- 3.3.1 Introductory Remarks.- 3.3.2 Procedure.- 3.4 Preparing, Purifying, and Storing a Gold Probe.- 3.4.1 General Remarks.- 3.4.2 The Preparation of a Polyclonal Antibody/Gold Probe for Use in Electron Microscopic Immunocytochemistry.- 3.4.3 The Preparation of a Monoclonal Antibody/Gold Probe for Use in Electron Microscopic Immunocytochemistry.- 3.4.4 The Preparation of Streptavidin/Gold and Protein A/Gold Probes for Use in Electron Microscopy.- 3.4.5 Use of Carbowax 20 M as Stabilizer.- 4 Quality Control and Analysis of Gold Sols and Probes.- 5 Critical Evaluation of the Use of Gold Probes in Selected Marking Techniques.- 5.1 Influence of the Size of the Gold Particles on Marking Efficiency.- 5.2 Gold Marking of Surface Components of Cells in Suspension and Monolayers.- 5.3 EM Localization of Targets in Tissues.- 5.3.1 General Remarks.- 5.3.2 Immunomarking of Ultrathin Sections of Resin-Embedded Tissues or Cells.- 5.3.3 Immunomarking of Thawed, Ultrathin Frozen Sections of Tissues or Cells.- 5.3.4 Protein A/Gold or Secondary Antibody/Gold Probes?.- 5.3.5 Double on-Grid Marking.- 5.4 Pre-Embedding Marking of Intracellular Targets in Cultured Cell Monolayers.- 6 Conclusions.- References.