Cryotechniques in biological electron microscopy

Abstract

I Fundamentals.- 1 Physics of Water and Ice: Implications for Cryofixation.- 1 Introduction.- 2 Functions of Water in Cryospecimens.- 3 Water Below Room Temperature.- 4 Aqueous Solutions Below Room Temperature.- 5 Specimen Cooling.- References.- 2 The Response of Biological Macromolecules and Supramolecular Structures to the Physics of Specimen Cryopreparation.- 1 Introduction.- 2 The Hydration Shells of Biological Macromolecules and Supramolecular Structures.- 3 Phases and Interface Phenomena.- 4 The Particular Case of the Biological Membrane.- 5 A Potpourri of Collapses.- References.- 3 Electron Beam Radiation Damage to Organic and Biological Cryospecimens.- 1 Introduction.- 2 Electron Beam/Specimen Interaction.- 3 Radiation Damage to Organic Materials at Low Temperature.- 4 Radiation Damage to Ice.- 5 Radiation Damage to Frozen-Hydrated and Vitrified-Hydrated Specimens.- 6 Conclusions.- References.- II General Methodology.- 4 Cryofixation Without Pretreatment at Ambient Pressure.- 1 Introduction.- 2 Cryofixation with Liquid Cryogen.- 3 Impact Cryofixation (Slamming).- 4 Discussion.- References.- 5 Cryoeleetron Microscopy of Vitrified Specimens.- 1 Introduction.- 2 Vitrification.- 3 Preparation of Thin Specimens.- 4 Image Formation.- 5 Beam Damage.- References.- 6 Cryoultramicrotomy.- 1 Introduction.- 2 Technical Aspects of Cryoultramicrotomy.- 3 Physical Aspects of Cryoultramicrotomy.- 4 Conclusions.- References.- 7 Freeze-Substitution and Freeze-Drying.- 1 Introduction.- 2 Methodology: Theoretical and Experimental Data.- 3 Procedures and Instrumentation.- 4 Critical Evaluation.- References.- III Special Aspects.- 8 Theory and Practice of High Pressure Freezing.- 1 Introduction.- 2 Freezing Under Atmospheric Pressure.- 3 Freezing Under High Pressure.- 4 The Main Practical Problems of Pressure-Freezing.- 5 The High Pressure Freezing Machine (Balzers HPM 010).- 6 Some Practical Advice.- 7 Discussion of Results.- References.- 9 Freeze-Etching of Dispersions, Emulsions and Macromolecular Solutions of Biological Interest 192.- 1 Introduction.- 2 Specific Problems of Specimen Preparation.- 3 Determination of Particle Concentrations and Molecular Weights.- 4 Measurements of Size and Shape.- 5 Structure of Dispersions in Bulk and at Interfaces.- References.- 10 High Resolution Metal Replication of Freeze-Dried Specimens.- 1 Introduction.- 2 Electron Microscopy and Image Processing.- 3 Characterization of the Test Specimens.- 4 Controlled Freeze-Drying.- 5 High Resolution Shadowing.- References.- 11 Immunogold Labelling of Cryosections and Cryofractures.- 1 Introduction.- 2 Cryoultramicrotomy.- 3 Cryofractures.- 4 Label Efficiency.- 5 Conclusions.- References.- 12 Cryoultramicrotomy for Autoradiography and Enzyme Cytochemistry.- 1 Introduction.- 2 Cryoultramicrotomy for the Autoradiography of Diffusible Substances.- 3 Thin Cryosections in Histochemistry.- 4 Conclusions.- References.- 13 Scanning Electron Microscopy and X-Ray Microanalysis of Frozen-Hydrated Bulk Samples.- 1 Introduction.- 2 Morphology.- 3 Analysis.- 4 Freezing.- 5 Instrumentation.- 6 Electron Interactions.- 7 X-Ray Emission.- 8 The Fracture Surface.- 9 Quantitative Analysis.- 10 Water Content or Dry Weight Fraction.- 11 Beam Damage and Mass Loss.- 12 Detection Limits.- 13 Conclusions.- References.- 14 Cryofixation of Dynamic Processes in Cells and Organelles.- 1 Introduction.- 2 Cooling Rate, Freezing Time, Time Resolution.- 3 Cellular Structures: Rapid Freezing Versus Chemical Fixation.- 4 Membrane Dynamics.- 5 Conclusions and Outlook.- References.- 15 Cryofixation of Diffusible Elements in Cells and Tissues for Electron Probe Microanalysis.- 1 Introduction.- 2 Specimen Preparation.- 3 Some Biological Applications and Results.- 4 Conclusion and Outlook.- References.- IV Appendix.- 16 Safety Rules for Cryopreparation.- 1 Introduction.- 2 Asphyxiation with Gaseous Nitrogen.- 3 Gaseous Propane Explosions.- 4 Burns Caused by Secondary Cryogen Splashing.- 5 Burns Caused by Primary Cryogen Splashing.- 6 Ignition of Combustible Secondary Cryogens.- 7 Bursting of Cryogen Containers.- 8 Transport and Disposal of Cryogens.- 9 Concluding Remarks.- References.