Advanced Techniques in Biological Electron Microscopy III

This volume is a continuation of two prior books on advanced electron microscope techniques. The purpose of this series has been to provide in­ depth analyses of methods which are considered to be at the leading edge of electron microscopic research procedures with applications in the biological sciences. The mission of the present volume remains that of a source book for the research practitioner or advanced student, especially one already well versed in basic electron optical methods. It is not meant to provide in­ troductory material, nor can this modest volume hope to cover the entire spectrum of advanced technology now available in electron microscopy. In the past decade, computers have found their way into many research laboratories thanks to the enormous increase in computing power and stor­ age available at a modest cost. The ultrastructural area has also benefited from this expansion in a number of ways which will be illustrated in this volume. Half of the contributions discuss technologies that either directly or indirectly make extensive use of computer methods.

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 Mülpordt.- 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.