Sample Preparation Handbook for Transmission Electron Microscopy : Methodology

Successful transmission electron microscopy in all of its manifestations depends on the quality of the specimens examined. Biological specimen preparation protocols have usually been more rigorous and time consuming than those in the physical sciences. For this reason, there has been a wealth of scienti?c literature detailing speci?c preparation steps and numerous excellent books on the preparation of b- logical thin specimens. This does not mean to imply that physical science specimen preparation is trivial. For the most part, most physical science thin specimen pre- ration protocols can be executed in a matter of a few hours using straightforward steps. Over the years, there has been a steady stream of papers written on various aspects of preparing thin specimens from bulk materials. However, aside from s- eral seminal textbooks and a series of book compilations produced by the Material Research Society in the 1990s, no recent comprehensive books on thin spe- men preparation have appeared until this present work, ?rst in French and now in English. Everyone knows that the data needed to solve a problem quickly are more imp- tant than ever. A modern TEM laboratory with supporting SEMs, light microscopes, analytical spectrometers, computers, and specimen preparation equipment is an investment of several million US dollars. Fifty years ago, electropolishing, chemical polishing, and replication methods were the principal specimen preparation me- ods. Cover Sample Preparation Handbook for Transmission Electron Microscopy Foreword Preface to the English Edition About the Authors Contents Abbreviations Chapter 1 Methodology: General Introduction Chapter 2 Introduction to Materials Introduction 1.1 Origin of Materials 1.2 Evolution of Materials 1.3 General Problems Presented by Microstructure Investigations Classification of Materials and Properties 2.1 Types of Chemical Bonds: Atomic and Molecular 2.2 Type of Materials and Chemical Bonds 2.3 Chemical Bonds and Mechanical Properties 2.3.1 Mechanical Properties and Crystallinity 2.3.2 Rigidity: From Hard to Soft 2.3.3 Tensile Strength: Ductility--Brittleness 2.3.4 Mechanical Properties of Organic Materials and Glass Transition ( T g ) Microstructures in Materials Science 3.1 Problems to Be Solved in Materials Science 3.2 Materials Microstructures 3.3 Polymer Microstructures 3.4 Crystalline Defects and Properties of Materials 3.5 Solid-State Polymer Properties Microstructures in Biological Materials 4.1 Problems to Be Solved in Biology 4.2 Singularity of Biological Materials: Importance of the Liquid Phase 4.3 Microstructure in Biology 4.4 Role of Structures on Functional Properties Bibliography Chapter 3 The Different Observation Modes in Electron Microscopy (SEM, TEM, STEM) Introduction Signals Used for Electron Microscopy 2.1 Electron--Matter Interaction 2.2 Signals Used for Imaging 2.3 Signals Used for Chemical Analysis 2.4 Signals Used for Structure 2.4.1 Transmitted Electrons: Thin Samples with Thickness 100 nm Microscopes and Observation Modes 3.1 Illumination Sources 3.1.1 Thermionic Sources 3.1.2 Field Emission Guns (FEGs) 3.2 Illumination Modes and Detection Limits 3.3 Microscope Resolutions and Analysis 3.3.1 Resolution Limit of the TEM 3.3.2 Spatial Resolution The Different Types of Microscopes: SEM, TEM, and STEM 4.1 Scanning Electron Microscope (SEM) 4.2 Conventional Transmission Electron Microscope (CTEM) 4.3 Analytical TEM/STEM Microscope and ''Dedicated STEM'' Different TEM Observation Modes 5.1 Origin of Contrast 5.1.1 Amplitude Contrast and Phase Contrast 5.2 Diffraction Contrast Imaging Modes in TEM and TEM/STEM 5.3 Chemical Contrast Imaging Modes in TEM and TEM/STEM 5.4 Spectroscopic Contrast Imaging Modes in TEM and TEM/STEM 5.5 EDS Chemical Analysis Methods in TEM and TEM/STEM 5.6 EELS Spectroscopic Analysis Modes in TEM and TEM/STEM Conclusion and Information Assessment Bibliography Chapter 4 Materials Problems and Approaches for TEMand TEM/STEM Analyses Introduction Analyses Conducted Prior to TEM Analyses 2.1 Macroscopic Characterization 2.2 Microscopic Characterization 2.3 Microscopic and Nanoscopic Characterization Approach for Beginning the Investigation of a Material Selection of the Type of TEM Analysis Analysis of Topography Structural Analysis in TEM 6.1 Morphology and Structure of Materials 6.2 Atomic Structure Crystallographic Analysis Analysis of Crystal Defects: 1D (Dislocations), 2D (Grain Boundaries and Interfaces), and 3D (Precipitates) EDS Chemical Analysis and EELS Spectroscopic Analysis 9.1 Phase Identification and Distribution 9.2 Concentration Profiles and Interface Analysis Structural Analyses Under Special Conditions 10.1 In Situ Analyses 10.1.1 At Room Temperature 10.1.2 At High Temperatures 10.1.3 At Low Temperatures 10.2 Cryomicroscopy 10.2.1 Structure of Isolated Particles from Biological Materials or Polymers 10.2.2 Structure of Bulk Frozen Samples Study of Properties 11.1 Optical Properties 11.2 Electrical Properties 11.3 Electronic Properties 11.4 Magnetic Properties 11.5 Mechanical Properties 11.6 Chemical Properties 11.7 Functional Properties Relationship Between Sample Thickness and Analysis Type in TEM and TEM/STEM Assessment of TEM Analyses Chapter 5 Physical and Chemical Mechanisms of Preparation Techniques Introduction Mechanical Action 2.1 Principles of a Material's Mechanical Behavior 2.2 Abrasion Principle 2.2.1 Techniques Involving Cutting by Means of Mechanical Abrasion: Sawing and Grinding 2.2.2 Abrasive Techniques: Mechanical Polishing, Dimpling, and Tripod Polishing 2.3 Rupture Principles 2.3.1 Techniques Involving Fracture: Crushing, Wedge Cleavage, Ultramicrotomy, and Freeze Fracture Chemical Action 3.1 Principle of Chemical and Electrochemical Dissolution 3.1.1 Techniques Involving Chemical and Electrochemical Dissolution Ionic Action 4.1 Ionic Abrasion Principles 4.2 Techniques Involving Ion Abrasion 4.2.1 Ion Beam Thinning and Focused Ion Beam Thinning (FIB) Actions Resulting in a State Change of Materials Containing an Aqueous Phase 5.1 Elimination of the Aqueous Phase 5.2 Freezing Principles 5.3 Principle of Substitution, Infiltration, and Embedding in Cryogenic Mode 5.4 Cryo-sublimation (or Freeze-Drying) Principle Actions Resulting in a Change in Material Properties 6.1 Chemical Fixation Principles 6.1.1 Constancy of pH 6.1.2 Molar Concentration 6.1.3 Ionic Concentration 6.2 Dehydration Principles 6.3 Infiltration Principles 6.4 Embedding or Inclusion Principles 6.5 ''Positive-Staining'' Contrast Principles Physical Actions Resulting in Deposition 7.1 Physical Deposition 7.2 Physics of the Coating Process 7.2.1 Nature of Chemical Elements Used as Sources 7.2.2 Different Methods of Particle Production 7.2.3 Vacuum 7.2.4 Substrate 7.3 Techniques Involving a Physical Deposition: Continuous or Holey Thin Film, Contrast Enhancement by Shadowing or Decoration, Replicas, and Freeze Fracture 7.3.1 Replica Techniques 7.3.2 Contrast Enhancement by Physical Coating: ''Negative-Staining'' Contrast Bibliography Mechanical Action Chemical Action Ionic Action Actions Resulting in a State Change of Materials Containing an Aqueous Phase Actions Resulting in a Change in Material Properties Physical Actions Resulting in a Deposit Chapter 6 Artifacts in Transmission Electron Microscopy Introduction Preparation-Induced Artifacts 2.1 Mechanical Preparation-Induced Artifacts 2.1.1 Secondary Thermal Damage Induced During Mechanical Preparation 2.2 Ionic Preparation-Induced Artifacts 2.2.1 Secondary Thermal Damage Induced During Ionic Preparation 2.3 Chemical Preparation-Induced Artifacts 2.3.1 Changes Specific to Biological Materials 2.3.2 Secondary Thermal Damage Induced During Chemical Preparation 2.4 Physical Preparation-Induced Artifacts 2.4.1 Secondary Thermal Damage Induced During Physical Preparation Artifacts Induced During TEM Observation 3.1 Artifacts Not Linked to Thermal Damages 3.2 Secondary Thermal Damage Examples of Artifacts 4.1 Artifacts Induced by the Tripod Polishing Technique 4.2 Artifacts Induced by the Ultramicrotomy Technique 4.3 Artifacts Induced by the Freeze-Fracture Technique 4.4 Artifacts Induced by Ion Milling or FIB 4.5 Artifacts Induced by the Substitution--Infiltration--Embedding Technique 4.6 Artifacts Induced by Chemical Fixation 4.7 Artifacts Induced by the Extractive-Replica Technique 4.8 Artifacts Induced by the Shadowing Technique 4.9 Artifacts Induced by the ''Positive-Staining'' Contrast Technique 4.10 Artifacts Induced by the Cryofixation Technique 4.11 Artifacts Induced by the Fine Particle Dispersion Technique 4.12 Artifacts Induced by the Frozen-Hydrated-Film Technique 4.13 Artifacts Induced by the ''Negative-Staining'' Contrast Technique 4.14 Artifacts Induced by the Electron Beam Summary Tables Bibliography Chapter 7 Selection of Preparation Techniques Based on Material Problems and TEM Analyses Introduction Classification of Preparation Techniques Characteristics of Preparation Techniques Criteria Used to Select a Preparation Technique Selection Criteria Based on Material Type Selection Criteria Based on Material Organization 6.1 Bulk Materials 6.2 Single-Layer or Multilayer Materials 6.3 Fine Particles Selection Criteria Based on Material Properties 7.1 Based on the Physical State of the Material 7.2 Based on the Chemical Phases in the Material 7.3 Based on the Electrical Properties of the Material 7.4 Based on the Mechanical Properties of the Material 7.4.1 Materials in Solid-State Physics 7.4.2 Soft-Ductile Materials 7.4.3 Hard-Resistant Materials 7.4.4 Materials of Intermediate Hardness and Ductility 7.4.5 Biological Materials Selection Criteria Related to the Type of TEM Analysis 8.1 Preparation Techniques Selection of the Orientation of the Sample Section 9.1 Microstructure Geometry 9.2 Defect Geometry Selection Criteria Related to Artifacts Induced by the Preparation Technique Adaptation of the Technique Based on Problems Related to Observation 11.1 Reducing Sample Thickness 11.2 Increasing Contrast 11.3 Reducing Charge Effects 11.4 Limitation of Strain Hardening 11.5 Removal of Surface Amorphization 11.6 Removal of Surface Contamination 11.7 Final Cleaning of the Thin Slice Conclusion Bibliography Chapter 8 Comparisons of Techniques Introduction Examples Using Fine Particle Materials 2.1 Comparison of Mechanical Preparations and Replicas 2.1.1 Crushing Technique (0Techniques0 Chapter 4 , Section 1 ) and Extractive Replica Technique (0Techniques0 Chapter 5 , Section 3 ) 2.1.2 Crushing Technique (0Techniques0 Chapter 4 , Section 1 ) and Extractive Replica Technique (0Techniques0 Chapter 5 , Section 3 ) 2.2 Comparison of ''Negative-Staining'' Contrast and Freeze-Fracture Techniques 2.2.1 Negative-Staining (0Techniques0 Chapter 7 , Section 2 ) and Freeze-Fracture Techniques (0Techniques0 Chapter 5 , Section 4) 2.3 Comparison of ''Negative-Staining'' and Decoration-Shadowing Contrast Techniques 2.3.1 Negative-Staining Contrast (0Techniques0 Chapter 7 , Section 2 ) and Decoration-Shadowing (0Techniques0 Chapter 7 , Section 1 ) Techniques 2.3.2 Negative-Staining (0Techniques0 Chapter 7 , Section 2 ) and Decoration-Shadowing (0Techniques0 Chapter 7 , Section 1 ) Contrast Techniques 2.4 Comparison of ''Positive-Staining'' and Decoration-Shadowing Contrast Techniques 2.4.1 Positive-Staining (0Techniques0 Chapter 7 , Section 2 ) and Decoration-Shadowing (0Techniques0 Chapter 7 , Section 1 ) Contrast Techniques Examples Using Bulk or Multilayer Materials 3.1 Comparison Between Different Mechanical Preparations 3.1.0 Wedge Cleavage Technique (0Techniques0 Chapter 4 , Section 2 ) and Tripod Polishing Technique (0Techniques0 Chapter 5 , Section 3 ) 3.2 Comparison Between Mechanical Preparations and Ionic Preparations 3.2.1 Comparison of ''Cleaved Wedge'' and ''Ionic Thinning'' Techniques 3.2.2 Comparison of ''Tripod Polishing + Ions'' and ''FIB Thinning'' 3.2.3 Comparison of ''Ultramicrotomy'' and ''Ion Milling'' Techniques 3.2.4 Comparison of ''Tripod Polishing,'' ''Ion Milling,'' and ''FIB Thinning'' Techniques 3.2.5 Comparison of ''Tripod Polishing'' and ''Tripod Polishing + Ion Milling'' Techniques 3.2.6 Comparison of ''Tripod Polishing'' and ''Ion Milling'' Techniques 3.3 Comparison Between Mechanical Preparations and Electrolytic Preparations 3.3.0 Electrolytic Thinning Techniques (0Techniques0 Chapter 3 , Section 1 ), Tripod Polishing Technique + Ion Milling Technique (0Techniques0 Chapter 4 , Section 3 ; 0Techniques0 Chapter 3 , Section 5), and Ultramicrotomy Technique (0Techniques0 Chapter 4 , Section 4) 3.4 Comparison Between Techniques Specific to Biology 3.4.1 Comparison of ''Chemical Methods,'' ''Physical Methods,'' and ''Freeze-Fracture'' Techniques 3.4.2 Comparison of ''Chemical Methods,'' ''Physical Methods,'' and ''Freeze-Fracture'' Techniques 3.4.3 Comparison of ''Chemical Fixation,'' ''Cryo-embedding,'' and ''Immunolabeling'' Techniques 3.4.4 Comparison of ''Immunolabeling,'' ''Ultramicrotomy,'' and ''Cryo-ultramicrotomy'' Techniques 3.5 Comparison Between All Techniques That Can Be Used in Biology on One Example: Collagen 3.5.1 Comparison of ''Negative-Staining Contrast'' and ''Immunolabeling techiques'' 3.5.2 Comparison of ''Negative-Staining'' and ''Decoration-Shadowing'' Contrast, and ''Freeze-Fracture'' Techniques 3.5.3 Comparison of ''Chemical Fixation,'' ''Physical Fixation,'' and ''Cryo-embedding'' Techniques Bibliography Chapter 9 Conclusion: What Is a Good Sample? Photo Credits Index This two-volume Handbook is a comprehensive and authoritative guide to sample preparation for the transmission electron microscope. This first volume covers general theoretical and practical aspects of the methodologies used for TEM analysis and observation of any sample. The information will help you to choose the best preparative technique for your application taking into account material types, structures, and their properties. Physical properties, material classification, and microstructures are considered together with a thorough description of the physics and chemistry of sample preparation and the main artifacts brought about by mechanical, physical and chemical methods, principles which are also applicable to sample preparation for the SEM, AFM etc ... Also included is a discussion of how to combine techniques for complex sample analysis and to obtain a TEM thin slice. Sample Preparation Handbook for Transmission Electron Microscopy: Methodology will guide you through the most current techniques for successful sample preparation in all fields from materials science to biology