Advanced Techniques in Biological Electron Microscopy

Embedding Media — Old and New.- A. Introduction.- B. Early Embedding Media.- I. Gelatin.- II. Celloidin.- III. Paraffin.- IV. Methacrylate.- C. Conventional Embedding Media.- I. Polyester Resins.- II. Epoxy Resins.- III. Water-soluble Embedding Media.- D. Advantages and Disadvantages of Conventional Embedding Media.- I. Polymerization Damage.- II. Beam Damage.- III. Erratic Polymerization.- IV. Shrinkage.- V. Viscosity.- VI. Osmotic Damage.- VII. Toxicity.- VIII. Miscellaneous Defects.- E. New Embedding Media.- I. Low Viscosity Epoxy Resins.- II. Exotic Embedding Materials.- 1. Hydrophilic Gels.- 2. Polyurethane Resins.- F. Conclusion.- References.- Substitution Techniques.- A. Introduction.- B. Inert-dehydration.- I. Method.- II. Artifacts.- III. Fine Structure.- C. Freeze-substitution.- I. Experiments of this Author.- II. The Work of Fernàndez-Moràn and Bullivant.- III. The Experiments of Rebhun and Associates.- IV. Experiments of Van Harreveld, Crowell and Malhotra.- V. Pertinent Findings of Other Investigators.- D. Conclusions.- References.- Freeze-Etching and Freeze-Fracturing.- A. Introduction.- B. Freezing of Biological Systems.- C. Methods and Instrumentation.- I. Historical Development.- II. Physical Basis of Technique.- 1. Fracturing.- 2. Etching.- 3. Replicating.- 4. Cleaning.- III. A Simple Freeze-Fracture Device.- 1. Pre-Treatment.- 2. Freezing.- 3. Fracturing.- 4. Replication.- 5. Cleaning of Replica.- IV. A Microtome Freeze-etch Device.- 1. Freezing.- 2. Fracturing.- 3. Etching.- 4. Replication.- 5. Cleaning of Replica.- V. Other Simple Devices.- 1. Geymeyer.- 2. Winkelmann.- 3. Weinstein.- 4. McAlear.- VI. Other Microtome Devices.- Koehler.- Steere.- Preston.- Edwards.- VII. Complementary Replicas.- 1. Chalcroft.- 2. Steere.- 3. Wehrli.- 4. Sleytr.- 5. Winkelmann.- VIII. Technical Variations.- 1. Pretreatment.- 2. Freezing.- 3. Replication.- D. Interpretation.- I. The Membrane Fracture Face.- 1. Complementary Replicas.- 2. Surface Labelling.- 3. Thin Sectioning.- II. Particles in Membranes.- 1. Lack of B Face Pits.- 2. The Nature of the Particles.- III. Contamination.- 1. Particulate Contamination.- 2. Plaque Contamination.- E. Conclusions.- I. Choice of Equipment.- II. Future.- References.- Electron Microscope Autoradiography.- F. A. McHenry.- A. Introduction.- B. Distribution of Developed Grains Around Radioactive Sources.- C. Analysis of Autoradiograms.- I. Qualitative Assessment.- II. Quantitative Analysis.- 1. “Simple Grain Density” Analyses.- 2. “Per cent” Analysis.- 3. “Probability Circle” Analysis.- 4. “Density Distribution” Analysis.- D. Conversion of Developed Grain Data to Information on Radioactivity.- References.- Scanning Electron Microscope Techniques in Biology.- A. Introduction.- I. General Principles of Operation.- II. A Comparison of Resolution.- III. Comparison of Information Transfer.- 1. Analytic Information.- 2. Subjective or Experiential Information Transfer.- B. Specimen Preparation.- I. Selection of Tissue.- 1. Natural Surfaces.- 2. Dissected Material.- 3. Sectioned Tissue.- 4. Living Specimens.- 5. Ion Etching.- 6. Freeze-Etching Techniques.- II. Fixation.- 1. Ultrastructure Fixatives.- 2. Light Microscope Fixatives.- III. Dehydration and Drying.- 1. Freeze-Drying.- 2. Critical Point Drying.- 3. Air Drying.- IV. Improving Conductivity.- 1. Metal Evaporation.- 2. Conducting Sprays and Solutions.- C. Viewing Techniques.- I. Standard Specimens.- II. Signal Monitor.- III. Accelerating Voltage.- IV. Specimen Current.- V. Contrast; Photo-multiplier.- VI. Scan Rate.- VII. Astigmatism Correction.- VIII. Final Aperture Size.- IX. Viewing Aspect.- X. Micromanipulation.- D. Signal Processing.- I. Differentiation.- II. Deflection Modulation.- III. Color Modulation.- IV. Computer Processing.- E. Recording Techniques.- I. Photographic Integration.- 1. Polaroid Film.- 2. 35 Millimeter Standard Roll Film.- II. Stereo-Pairs.- 1. Resolution of Analytic Ambiguities.- 2. Enhancement of Experiential Contact.- 3. Methods of Stereo-Pair Presentation.- III. TV Tape.- F. Information Assimilation by the Observer.- I. Analytic Information Processing.- 1. Geometric Information.- a) Metric Geometry.- b) Topologic Geometry.- 2. Chemical Information.- a) Characteristic X-Ray Elemental Analysis.- b) Auger Spectra.- c) Cathodoluminescene Analysis.- d) Enery-loss Spectra.- 3. Electrical Properties and Charging.- II. Experiential or Subjective Information Processing....- 1. Models of Perception.- 2. Limits of Analytical Information Processing…..- 3. Possibilities of Complementary Subjective and Analytic Investigations.- G. Conclusion.- I. Questions Regarding a Scanning Electron Microscope Program for Biological Study.- 1. Is the SEM Really Necessary?.- 2. Which Instrument?.- 3. What Auxilliary Equipment Might be Needed?.- II. Prospects for the Future.- H. Appendices.- I. Optical Aids for the Viewing of Vertically Mounted Stereo-Pairs.- II. Projection of Stereo-Pairs by Means of a Superimposed Color-Coded Transparency.- References.- Computer Processing of Electron Micrographs.- A. Introduction.- B. Linear Systems and Fourier Processing.- I. The Concept of Linear Systems.- II. Fourier Integrals and Theorems.- III. Implementation on the Computer.- C. Digitizing of Electron Micrographs.- I. Photographic Recording.- II. The Densitometer.- III. Sampling.- IV. The Effect of the Scanning Aperture.- V. The Effect of the Image Boundary.- D. Noise Filtering.- I. Noise Sources.- II. Noise Filtering in the Case of Periodic Objects.- III. Noise Filtering in the Case of Aperiodic Objects…..- E. The Cross Correlation Function and its Use for Image Alignment.- I. Two Electron Micrographs with Identical Defocus Value.- II. Two Electron Micrographs with Different Defocus Values.- III. A Technical Note.- F. Two-Dimensional Restoration.- I. Restoration of Phase Objects from a Single Phase Contrast Image.- II. Restoration of Phase Objects from a Focus Series…..- III. Restoration of the Complex Object.- IV. Restoration from Dark Field Images.- G. Object/Support Separation.- I. Optimal Filtering.- II. Matched Filtering.- III. Separation Based on Knowledge of the Film Structure.- IV. Separation Based on the Z-dependence of the Imaginary Scattering.- H. Three-Dimensional Reconstruction.- I. The Fourier Method.- 1. Principle of the Three Dimensional Fourier Reconstruction.- 2. The Interpolation Problem.- 3. The Use of Symmetries.- 4. Implementation.- 5. A Two-dimensional Fourier Reconstruction Scheme.- I. Real Space Methods.- 1. Exact Solution.- 2. Superposition Method.- 3. Iterative Approximation.- References.- High Voltage Electron Microscppy.- A. Introduction.- B. Merits of the High Voltage Electron Microscope.- I. Specimen Penetration.- II. Resolving Power.- III. Beam Damage.- C. Biological Applications.- I. Specimen Preparation.- II. High Resolution Observation.- III. Observation of Thick Specimens.- IV. High Voltage Stereoscopy.- V. Observation of Undehydrated Specimens.- 1. Ultracryotome Method.- 2. Wet Cell Method.- D. Design and Construction of High Voltage Electron Microscopes.- References.