Mitochondrial Medicine: Volume 3: Manipulating Mitochondria and Disease- Specific Approaches

Preface
Contents
Contributors
Chapter 1: Allotopic Expression of ATP6 in Mouse as a Transgenic Model of Mitochondrial Disease
	1 Introduction
	2 Materials
		2.1 Construct Design
		2.2 Gene Synthesis
		2.3 Assembly of Transgene Expression Constructs
		2.4 Animal Genotyping
	3 Methods
		3.1 Construct Design
		3.2 Gene Synthesis and Construct Assembly (See Note 7)
		3.3 Generation of Founder Animals
		3.4 Animal Genotyping
	4 Notes
	References
Chapter 2: Mitochondrial Transplantation for Ischemia Reperfusion Injury
	1 Introduction
	2 Mitochondrial Transplantation Techniques
		2.1 Direct Epicardial Injection
			2.1.1 Materials
			2.1.2 Method
			2.1.3 Pros
			2.1.4 Cons
		2.2 Intracoronary Delivery
			2.2.1 Materials
			2.2.2 Method
			2.2.3 Pros
			2.2.4 Cons
	3 Co-incubation
		3.1 Materials
		3.2 Method
			3.2.1 Cell Plating
			3.2.2 Isolate Mitochondria
			3.2.3 For Mitochondrial Uptake
			3.2.4 For ATP or Other
			3.2.5 Cells
			3.2.6 Pros
			3.2.7 Cons
	4 Discussion
		4.1 Mitochondria Isolation
		4.2 Mitochondrial Source
		4.3 Suspension in Solution
		4.4 Stability of Isolated Mitochondria
		4.5 Mitochondrial Concentration
		4.6 Safety
		4.7 Autologous and Heterologous Mitochondria
		4.8 Mitochondrial Uptake and Internalization
		4.9 Endocytosis
		4.10 Ca2+ and Mitochondrial Function
		4.11 Mechanism of Action
		4.12 Other  Uses
	References
Chapter 3: Quantitatively Controlled Intercellular Mitochondrial Transfer by Cell Fusion-Based Method Using a Microfluidic Dev...
	1 Introduction
	2 Materials
		2.1 Equipment for Microfluidic Device Fabrication
		2.2 Microfluidic Device Components
		2.3 Cell Fusion (Mitochondrial Transfer) Equipment
		2.4 Cell Fusion (Mitochondrial Transfer) Components
	3 Methods
		3.1 Master Mold Fabrication
		3.2 Microfluidic Device Fabrication
		3.3 Cell Fusion Through a Microtunnel (Mitochondrial Transfer)
	4 Notes
	References
Chapter 4: Lipophilic Conjugates for Carrier-Free Delivery of RNA Importable into Human Mitochondria
	1 Introduction
	2 Materials
		2.1 Synthesis of Hydrazide of 6-(Cholesteryloxycarbonylamino)Hexanoic Acid (See Note 1)
		2.2 Synthesis of Oligoribonucleotides and Their Aldehyde-Modified Analogs
		2.3 Synthesis of Oligoribonucleotides Conjugated with Cholesterol Through Hydrazone Bond
		2.4 Analysis and Purification of Lipophilic RNA Conjugates
		2.5 Transfection of Human Cultured Cells with Lipophilic Conjugates
	3 Methods
		3.1 Synthesis of Hydrazide of 6-(Cholesteryloxycarbonylamino)Hexanoic Acid
			3.1.1 6-(Cholesteryloxycarbonylamino)-Hexanoic Acid (Compound 1 Fig. 2, Step i))
			3.1.2 Methyl 6-(Cholesteryloxycarbonylamino)Hexanoate (Compound 2 Fig. 2, Step ii)
			3.1.3 Hydrazide of 6-(Cholesteryloxycarbonylamino)Hexanoic Acid (Compound 3 Fig. 2, Step iii)
		3.2 Automated Phosphoramidite Synthesis of Oligoribonucleotides and Their Aldehyde-Modified Analogs
		3.3 Solid-Phase Synthesis of Oligoribonucleotides Conjugated with Cholesterol Through Hydrazone Bond
			3.3.1 Conjugation of 5′-Aldehyde-Modified Polymer-Bound Protected RNAs with Hydrazide of 6-(Cholesteryloxycarbonylamino)Hexano...
			3.3.2 Cleavage of Lipophilic Conjugates of RNAs from the Support and Removal of Base-Labile and 2′-O-TBDMS Protecting Groups
			3.3.3 Analysis and Purification of Crude Lipophilic Conjugates of RNAs
		3.4 Synthesis ``In Solution´´ of Oligoribonucleotides and Their Fluorophore-Labeled Derivatives Conjugated with Cholesterol th...
			3.4.1 Cleavage of Aldehyde-Modified RNAs from the Support and Removal of Base-Labile and 2′-O-TBDMS Protecting Groups (Fig. 5)
			3.4.2 Conjugation of 5´-Aldehyde-Containing RNAs with Hydrazide of 6-(Cholesteryloxycarbonylamino)Hexanoic Acid ``in Solution´´
			3.4.3 Synthesis of Fluorophore Labeled Conjugates
		3.5 Transfection of Cultured Human Cells with Lipophilic Conjugates
	4 Notes
	References
Chapter 5: Drug Discovery Assay to Identify Modulators of the Mitochondrial Ca2+ Uniporter
	1 Introduction
	2 Materials
		2.1 Functional Reconstitution of the Uniporter Activity in Yeast Mitochondria
			2.1.1 Reagents/Stock Solutions
			2.1.2 Working Solutions
		2.2 Growth of Functionally Reconstituted Yeast Cells
			2.2.1 Reagents/Stock Solutions
			2.2.2 Working Solutions
		2.3 Isolation of Functionally Reconstituted Yeast Mitochondria for Drug Screening
			2.3.1 Reagents/Stock Solutions
			2.3.2 Working Solutions
		2.4 Yeast Mitochondria-Based Drug Screening Assay
			2.4.1 Reagents/Stock Solutions
			2.4.2 Working Solutions
	3 Equipment
		3.1 Functional Reconstitution of Uniporter Activity in Yeast Mitochondria
		3.2 Growth of Functionally Reconstituted Yeast Cells
		3.3 Isolation of Functionally Reconstituted Yeast Mitochondria for Drug Screening
		3.4 Yeast Mitochondria-Based Drug Screen
	4 Methods
		4.1 Functional Reconstitution of the Uniporter Activity in Yeast Mitochondria
		4.2 Subcellular Localization of Yeast Heterologous Proteins by SDS-PAGE and Western Blotting
		4.3 Growth of Functionally Reconstituted Yeast Cells
		4.4 Isolation of Functionally Reconstituted Yeast Mitochondria for Drug Screening
		4.5 Yeast Mitochondria-Based Drug Screen
	5 Notes
	References
Chapter 6: Cytoplasmic Transfer Methods for Studying the Segregation of Mitochondrial DNA in Mice
	1 Introduction
	2 Materials
		2.1 Embryo Collection
		2.2 Embryo Manipulation
	3 Methods
		3.1 Embryo Collection for Ooplasm Transfer
		3.2 Ooplasm Transfer
		3.3 Embryo Collection for Blastomere Fusion
		3.4 Blastomere Fusion Embryo Manipulation
		3.5 Blastomere Fusion of Reconstructed Embryos
		3.6 Embryo Culture and Transfer
	4 Notes
	References
Chapter 7: Electron Attachment to Isolated Molecules as a Probe to Understand Mitochondrial Reductive Processes
	1 Introduction
	2 Electron Transmission Spectroscopy
	3 Dissociative Electron Attachment Spectroscopy
	4 Computational Methods
	5 Likely Mechanism of Toxicity of Halogenated Species Against Mitochondria
	6 Hypothetical Mechanism of Beneficial Activity of Polyphenolic Antioxidants Inside Mitochondria
	7 Notes
	References
Chapter 8: Assessment of Short- and Medium-Chain Fatty Acids on Mitochondrial Function in Severe Inflammation
	1 Introduction
	2 Materials
	3 Methods
		3.1 Isolation and Culture of Human Umbilical Vein Endothelial Cells (HUVEC)
		3.2 Isolation and Culture of Human Monocytes
		3.3 Stimulation of HUVEC and Monocytes with Fatty Acids
		3.4 High-Resolution Respirometry
		3.5 Summary and Comments
	4 Notes
	References
Chapter 9: Assessment of the Effects of Drugs on Mitochondrial Respiration
	1 Introduction
	2 Materials
		2.1 Devices for Mitochondrial Respiration Measurements
		2.2 Media and Reagents
	3 Methods
		3.1 Isolation of Pig Brain Mitochondria
			3.1.1 Crude Mitochondrial Fraction (CMF)
			3.1.2 Mitochondria Purified on Sucrose Gradient (MPS)
		3.2 Effects of Drugs on Mitochondrial Respiration
			3.2.1 Protocol for the Drug Effect on Complex I-Linked Respiration
			3.2.2 Protocol for the Drug Effect on Complex II-Linked Respiration
			3.2.3 Protocol for Drug Effect on Complex IV-Linked Respiration
			3.2.4 Protocol for the Protective Effect of Drugs on Ca2+-Induced Change of the Complex I+II-Linked Respiration
		3.3 Data Analysis
	4 Notes
	References
Chapter 10: The NDUFS4 Knockout Mouse: A Dual Threat Model of Childhood Mitochondrial Disease and Normative Aging
	1 Introduction
	2 Materials
		2.1 Genotyping Supplies
		2.2 Reagents and Materials for Injections
		2.3 Special Diets and Medicated Water
		2.4 Tissue Collection
		2.5 Perfusions for Histology
		2.6 Anesthesia
	3 Methods
		3.1 Preparation and Storage of Rapamycin for Injection
		3.2 Special Diet Preparation
		3.3 Medicated Water Preparation
		3.4 Breeding
		3.5 Genotyping
		3.6 Weaning
		3.7 Common Drug Treatment Regimens
		3.8 End Point Criteria and Euthanasia
		3.9 Life Span Study Design
		3.10 Tissue Collection
		3.11 Perfusions
	4 Notes
	References
Chapter 11: Suborganellar Localization of Mitochondrial Proteins and Transcripts in Human Cells
	1 Introduction
	2 Materials
		2.1 Preparation of Human Cells and Their Transfection
		2.2 Cell Fixation, Permeabilization, and Immunolabeling
		2.3 Labeling of Mitochondrial RNA Granules by Bromouridine Inclusion into Newly Synthesized Transcripts
		2.4 Immunolabeling and Single-Molecule RNA In Situ Hybridization
		2.5 Conventional Confocal Microscopy: Image Acquisition, Processing and Colocalization Analysis
		2.6 dSTORM: Sample Preparation, Image Acquisition, and Processing
	3 Methods
		3.1 Preparation of Human Cells and Transfection
		3.2 Cell Fixation, Permeabilization, and Immunolabeling
		3.3 Labeling of Mitochondrial RNA Granules by Bromouridine Incorporation in Newly Synthesized Transcripts
		3.4 Immunolabeling and Single-Molecule RNA In Situ Hybridization
		3.5 Conventional Confocal Microscopy: Image Acquisition, Processing, and Colocalization Analysis
		3.6 3D dSTORM: Sample Preparation, Image Acquisition, and Processing
	4 Notes
	References
Chapter 12: In Vivo Assessment of Mitochondrial Oxygen Consumption
	1 Introduction
	2 Materials
		2.1 Laser Setup (Fig. 1)
		2.2 Animals
		2.3 Chemicals, Fluids, and Reagents
		2.4 Surgical Tools
		2.5 Ventilation and Monitoring
	3 Methods
		3.1 Delayed Luminescence Setup
		3.2 Animal Preparation (Fig. 3)
		3.3 Experimental Procedures
	4 Notes
	References
Chapter 13: Update of Mitochondrial Network Analysis by Imaging: Proof of Technique in Schizophrenia
	1 Introduction
	2 Materials
	3 Methods
		3.1 Cell Preparation
		3.2 Coating Imaging Plates (Under Sterile Conditions)
			3.2.1 PDL/PLL Plate Coating
			3.2.2 Matrigel Plate Coating
		3.3 JC-1 or MitoTracker Orange Staining Procedure
		3.4 Imaging Procedure
			3.4.1 Steady-State Imaging of Mitochondrial Deltaψm and Mitochondrial Intracellular Distribution
			3.4.2 Imaging Procedure for Assessing Mitochondrial Network Connectivity
		3.5 Imaging Analyses
			3.5.1 Analysis of Mitochondrial Deltaψm
			3.5.2 Analysis of Mitochondrial Network Morphology
			3.5.3 Analysis of Mitochondrial Network Connectivity
	4 Notes
	References
Chapter 14: Heterologous Inferential Analysis (HIA) and Other Emerging Concepts: In Understanding Mitochondrial Variation In P...
	1 Introduction
	2 HIA
	3 Variants in mt-tRNA Genes
	4 Conclusions and Future Perspectives
	References
Chapter 15: Accurate Measurement of Cellular and Cell-Free Circulating Mitochondrial DNA Content from Human Blood Samples Usin...
	Abbreviations
	1 Introduction
	2 Materials
		2.1 Blood Collection and Storage
			2.1.1 Collection of Whole Blood
			2.1.2 Separation of PBMCs and Plasma from Whole Blood
			2.1.3 Separation of Serum from Whole Blood
		2.2 DNA Extraction
			2.2.1 DNA Extraction from Whole Blood and PBMCs
			2.2.2 DNA Extraction from Plasma and Serum
		2.3 Dilution Standards Preparation
		2.4 qPCR
		2.5 Equipment
	3 Methods
		3.1 Collection and Storage of Samples
			3.1.1 Collection of Whole Blood
			3.1.2 Separation of PBMCs and Plasma from Whole Blood
			3.1.3 Separation of Serum from Whole Blood
		3.2 Extraction of DNA from Blood Samples
			3.2.1 DNA Isolation from Whole Blood and PBMCs
			3.2.2 DNA Isolation from Plasma and Serum
		3.3 Pretreatment/Fragmentation of DNA (See Note 5)
		3.4 Preparation of Standards for Absolute Quantification
		3.5 Real-Time Quantitative PCR
		3.6 How to Analyze Your Results and Check Specificity/Accuracy of the Experiment
		3.7 Calculation of the Sample Size to Adequately Power the Study
		3.8 Conclusion
	4 Notes
	References
Chapter 16: Measurement of Mitochondrial Respiration in Platelets
	1 Introduction
	2 Materials
		2.1 Media and Reagents
		2.2 Device for Mitochondrial Respiration Measurement
		2.3 Microsyringes
	3 Methods
		3.1 Platelet Preparation
		3.2 Polarographic Measurement of Respiration
			3.2.1 Mitochondrial Respiration in Intact Platelets
			3.2.2 Mitochondrial Respiration in Permeabilized Platelets
		3.3 Data Analysis
	4 Notes
	References
Chapter 17: Isolation and Electron Microscopic Analysis of Liver Cancer Cell Mitochondria
	1 Introduction
	2 Materials
		2.1 Isolation
		2.2 Density Gradient
		2.3 Electron Microscopy
	3 Methods
		3.1 Isolation of Crude Mitochondria
		3.2 Purification of Crude Mitochondria by Density Gradient Centrifugation (Figs. 2 and 3) (See Note 12)
		3.3 Fixation and Embedding of Mitochondria for Electron Microscopy (See Note 15)
		3.4 Ultra-Thin Sectioning, Contrasting, and Imaging
		3.5 Image Analysis
	4 Notes
	References
Chapter 18: Assessment of Mitochondrial Reactive Oxygen Species and Redox Regulation in Stem Cells
	1 Introduction
	2 Materials
		2.1 Labeling of Muscle Stem Cells
		2.2 MitoSOX Red Quantification
		2.3 Gene Expression Analysis of Redox Regulation
	3 Methods
		3.1 Labeling of Muscle Stem Cells
		3.2 MitoSOX Red Staining and Quantification
		3.3 Gene Expression Analysis to Assess Redox Regulation
	4 Notes
	References
Chapter 19: Single-Cell Approaches for Studying the Role of Mitochondrial DNA in Neurodegenerative Disease
	Abbreviations
	1 Introduction
	2 Mitochondrial DNA Encoding Content and its Maintenance
	3 MtDNA Copy Number and the Threshold Effect
	4 The Role of mtDNA Changes in Normal Aging and in Age-Related Neurodegenerative Disease
		4.1 Susceptibility of mtDNA in Parkinson´s Disease: A Growing Realization for Cell Type-Specific Variation
	5 Experimental Methods for Exploring Cell-Specific Brain Pathology
		5.1 Tissue Preparation, Positive Identification of Cell Types, and Cell Isolation for Downstream Applications
		5.2 Mitochondrial Copy Number Variation and Measurement of mtDNA Deletion Levels
	6 Conclusions and Future Directions
	References
Chapter 20: A New Method for Sequencing the Mitochondrial Genome by Using Long Read Technology
	1 Introduction
	2 Materials
		2.1 Equipment and Materials
		2.2 Sample Collection and Storage
		2.3 DNA Extraction
		2.4 Agarose Gel Electrophoresis
		2.5 Nuclear DNA Depletion
		2.6 Multiple Displacement Amplification
		2.7 Library Preparation
			2.7.1 Enzymes from new England Biolabs
			2.7.2 Consumables from Oxford Nanopore Technologies
			2.7.3 Other Consumables
	3 Methods
		3.1 Sample Collection and Storage
		3.2 DNA Extraction
		3.3 Nuclear DNA Depletion
		3.4 Primers for Multiple Displacement Amplification
		3.5 Multiple Displacement Amplification
		3.6 MinION Library Preparation
			3.6.1 Purify and Size Select the Input DNA Samples
			3.6.2 Debranching Treatment of the Size Selected  DNA
			3.6.3 Library Construction
			3.6.4 Barcode Ligation and Cleanup
			3.6.5 Adapter Ligation Using the NEBNext Quick Ligation Module and DNA Cleanup
		3.7 Sequencing Run on MinION Sequencer
	4 Notes
	References
Chapter 21: Mitochondrial DNA as a Sensitive Biomarker of UV-Induced Cellular Damage in Human Skin
	1 Introduction
	2 Materials
		2.1 DNA Extraction Components
		2.2 Real-Time qPCR Components
			2.2.1 For the 500 base pair (bp) mtDNA damage Assay 1
			2.2.2 For the alternative 500 bp mtDNA damage Assay 2
			2.2.3 For the 83 bp Assay
			2.2.4 For all Assays
		2.3 Gel Electrophoresis Components
	3 Methods
		3.1 DNA Extraction
		3.2 Real-Time  qPCR
		3.3 Gel Electrophoresis
	4 Notes
	References
Chapter 22: Isolation of Mitochondria-Associated ER Membranes (MAMs), Synaptic MAMs, and Glycosphingolipid Enriched Microdomai...
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Solutions Required for the Isolation of Crude Mitochondria
		2.3 Solutions Required for the Isolation of MAMs
		2.4 Solutions Required for the Isolation of GEMs
	3 Methods
		3.1 Purification and Isolation of Mitochondria and MAMs from Brain Tissue
			3.1.1 Removal of Nuclei, Intact Cells, and Cellular Debris
			3.1.2 Isolation of Crude Mitochondria
			3.1.3 Isolation of MAMs
			3.1.4 Isolation of MAMs from Synaptosomes
		3.2 Purification and Isolation of Mitochondria and MAMs from MEFs and Neurospheres
			3.2.1 Removal of Nuclei, Intact Cells, Cellular Debris, and Isolation of Crude Mitochondria
			3.2.2 Isolation of MAMs
		3.3 Extraction of GEMs from Mitochondria and MAMs Isolated from Brain Tissue and Cells
		3.4 Anticipated Results
	4 Notes
	References
Chapter 23: Mitochondrial Diagnostics: A Discovery-Based Biochemical Platform for Phenotyping Human Peripheral Blood Cell Mito...
	1 Introduction
	2 Materials
		2.1 Isolation of PBMCs from Whole Blood
		2.2 Isolation of Mitochondria from PBMCs
		2.3 Respirometry Control Measurements in Permeabilized Cells and Isolated Mitochondria from PBMCs
		2.4 Enzymatic Activity of Matrix Dehydrogenases
		2.5 Citrate Synthase Activity
	3 Methods
		3.1 Isolation of PBMCs from Whole Blood
		3.2 Preparation of Intact and Permeabilized PBMCs
		3.3 Mitochondrial isolation from PBMCs
		3.4 Measuring Respiratory Flux Using the Protonophore FCCP in Intact PBMCs
		3.5 Measuring Respiratory Flux Using Creatine Kinase Clamp in Permeabilized PBMCs
		3.6 Measuring Respiratory Flux Using the Protonophore FCCP in Isolated Mitochondria from PBMCs
		3.7 Measuring Respiratory Flux Using the Creatine Kinase Clamp in Isolated Mitochondria
		3.8 Measuring Respiratory Flux and ATP Synthesis Supported by ADP Clamp in Isolated Mitochondria
		3.9 Enzymatic Activity of Matrix Dehydrogenases
		3.10 Citrate Synthase Activity Assay
	4 Notes
	References
Chapter 24: Bioenergetic Profiling of Human Pluripotent Stem Cells
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Reagents
		2.3 Reagent Setup
	3 Methods
		3.1 Growth of PSCs Under Feeder-Free Conditions on Matrigel-Coated Plates Prior to the Assay
		3.2 Day 1: Seeding PSCs onto a XF96 Cell Culture Microplate and Preparation of XF96 FluxPak
		3.3 Day 2: Exchange of Assay Medium, Loading of XFe 96 Sensor Cartridge with Injection Compounds and Assay  Run
		3.4 Enzymatic Assessment of Lactate Production Using Supernatant Media of the Cells
		3.5 Normalization by Measuring Cellular DNA Content and Data Analysis
	4 Notes
	References
Chapter 25: Integrative Methods for Studying Cardiac Energetics
	1 Introduction
		1.1 Energy Homeostasis in Heart
		1.2 Parallel Activation of Mitochondria and Myofibrils by Calcium
		1.3 Drug Effects: Levosimendan
		1.4 Comparison of the Effects of Levosimendan and Adrenaline
		1.5 Conclusion & Perspectives
	2 Methods and Rationale
		2.1 Methods
			2.1.1 Ethical Approval
			2.1.2 Heart Perfusion and 31P NMR Spectroscopy
			2.1.3 Myocardium Oxygen Consumption
		2.2 Modular Control Analysis of Heart Energetics (MoCA)
			2.2.1 Rationale and System Definition
			2.2.2 Experimental Determination of the Elasticities
			2.2.3 Regulation Analysis
	References
Chapter 26: Isolation of Mitochondria from Retinal Pigment Epithelial Cell Cultures and an Application of High-Resolution Resp...
	1 Introduction
	2 Materials
		2.1 Isolation of Mitochondria
		2.2 XFe96 High-Resolution Respirometric Assay
		2.3 Imaging for Mitochondrial Purity
	3 Methods
		3.1 Mitochondrial Isolation
		3.2 XFe96 High-Resolution Respirometric Assay
		3.3 Imaging for Mitochondrial Purity and Coverage on XFe96 Assay Plates
		3.4 Data Analysis
	4 Notes
	References
Chapter 27: The Isolation and Deep Sequencing of Mitochondrial DNA
	1 Introduction
	2 Materials
		2.1 COX/SDH Histochemical Staining (Skeletal Muscle Tissue)
		2.2 Laser Capture Microdissection (Single-Cells, Tissue)
		2.3 Long-Range PCR Enrichment of mtDNA (Tissue Homogenate and Grouped Cells)
		2.4 PCR Enrichment of mtDNA (Single-Cell Lysate)
		2.5 Sequencing Reagents for Illumina MiSeq
		2.6 Sequencing Data Analysis
	3 Methods
		3.1 COX/SDH Histochemical staining (Skeletal Muscle Tissue)
		3.2 Laser Capture Microdissection (Single Cells, Tissue)
		3.3 Long-Range PCR Enrichment of mtDNA (Tissue Homogenate and Grouped Cells)
		3.4 PCR Enrichment of mtDNA (Single-Cell Lysate)
		3.5 Sequencing Using Illumina MiSeq
		3.6 Sequencing Data Analysis
	4 Notes
	References
Chapter 28: Ultrastructure of the Mitochondria-Associated Membranes in Human Tumor Specimens
	1 Introduction
	2 Materials
	3 Methods
		3.1 Primary Fixation (See Note 2)
		3.2 Secondary Fixation (See Note 3)
		3.3 Dehydration (See Note 4)
		3.4 Infiltration and Embedding (See Note 5)
		3.5 Curing or Polymerization (See Note 6)
		3.6 Trim Block and Cut Thick Sections (See Note 7)
		3.7 Cut Thin Sections (See Note 8)
		3.8 Electronic Stain (See Note 9)
		3.9 Capturing Images with the Transmission Electron Microscope
	4 Notes
	References
Index