Advanced Techniques in Biological Electron Microscopy

The past decade has seen a remarkable increase in the use of electron microscopy as a researm tool in biology and medicine. Thus, most institu­ tions of higher learning now boast several electron optical laboratories having various levels of sophistication. Training in the routine use of elec­ tron optical equipment and interpretation of results is no longer restricted to a few prestigious centers. On the other hand, temniques utilized by researm workers in the ultrastructural domain have become extremely diverse and complex. Although a large number of quite excellent volumes of electron microscopic temnique are now dedicated to the basic elements available whim allow the novice to acquire a reasonable introduction to the field, relatively few books have been devoted to a discussion of more ad­ vanced temnical aspects of the art. It was with this view that the present volume was conceived as a handy reference for workers already having some background in the field, as an information source for those wishing to shift efforts into more promising temniques, or for use as an advanced course or seminar guide. Subject matter has been mosen particularly on the basis of pertinence to present researm activities in biological electron microscopy and emphasis has been given those areas whim seem destined to greatly expand in useful­ ness in the near future.

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.