Electron Microscopy : Principles and Techniques for Biologists

, by Bozzola, John J.; Russell, Lonnie D.

ISBN: 9780867201260 | 0867201266
Cover: Hardcover
Copyright: 3/1/1991

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Summary

Electron Microscopy covers all of the important aspects of electron microscopy for biologists, including theory of scanning and transmission, specimen preparation, digital imaging and image analysis, laboratory safety and interpretation of images. The text also contains a complete atlas of ultrastructure.

Preface

Acknowledgments

xvii

The Past, Present, and Future of Electron Microscopy

(12)

Historical Perspective

(3)

Development of the Electron Microscope

(2)

Development of Preparative Techniques

(1)

Contributions to Biology and the Future of Electron Microscopy

(1)

Journals Devoted Primarily to Electron Microscopy

(1)

Selected Journals Publishing Electron Micrographs

(1)

References

(3)

Specimen Preparation for Transmission Electron Microscopy

(26)

Fixation

(10)

The Mechanism of Chemical Fixation for Electron Microscopy

(1)

Glutaraldehyde

(2)

Osmium Tetroxide

(1)

Selection of a Fixative and a Buffer

(1)

Fixative

(1)

Buffers

(1)

Obtaining and Preparing Buffered Glutaraldehyde Fixative

(1)

Obtaining and Preparing Osmium Fixative

(1)

Immersion and Perfusion Fixation

(1)

Immersion

(1)

Perfusion Fixation

(1)

Fixation Conditions

(1)

Popular Fixation Protocols Other Than Glutaraldehyde---Osmium Tetroxide

(1)

Karnovsky's Fixative

(1)

Osmium-reduced Ferrocyanide

(1)

Potassium Permanganate

(1)

Fixative Additives

(1)

Fixation of Plant Tissues

(1)

Washing

(1)

Dehydration

(1)

Use of Transitional Solvents

(1)

Infiltration of Resin

(1)

Embedding

(2)

Epon Embedding

(1)

Measuring Embedding Media

(1)

Mixing Embedding Media

(1)

Other Embedments and Their Use

(1)

Epoxy Resin 812

(1)

Araldite

(1)

Spurr's

(1)

LR White

(1)

Low Pressure Removal of Solvents and Air Bubbles

(1)

Curing of the Embedment

(2)

Embedding Containers

(1)

Embedding Labels

(1)

Removal of Tissue Supports

(1)

Special Tissue Preparative Needs

(3)

Cell Fractions

(1)

Tissue Culture Cells

(2)

Suitable Containers for Tissue Processing

(1)

Rapid Tissue Processing Protocols

(1)

Automatic Tissue Processors

(1)

Tissue Volume Changes During Specimen Preparation

(1)

Judging Adequate Specimen Preparation

(1)

References

(3)

Specimen Preparation for Scanning Electron Microscopy

(24)

Surface Cleaning

(1)

How to Remove Surface Coatings

(1)

Buffers and Fixatives

(3)

Working with Individual Cells or Macromolecules

(1)

Rinsing and Dehydration

(1)

Specimen Drying Techniques

(4)

Critical Point Drying

(1)

Safety Precautions with Critical Point Drying

(1)

Freeze-Drying

(1)

Freeze-Drying Procedure

(1)

Alternative Drying Procedures

(1)

Fluorocarbon Drying Procedure

(1)

Specimen Fracturing Procedures

(3)

Cryofracturing

(1)

Cryofracturing Procedure of Tanaka

(2)

Dry Fracturing

(1)

Replication Procedures

(3)

Negative Surface Replication Using Cellulose Acetate Film

(1)

Cellulose Acetate Negative Replication Procedure

(1)

Negative/Positive Replication Using Silicone, Resin

(1)

Silicone, Resin Positive/Negative Replication Procedure

(1)

Corrosion Casting of Animal Vasculatures

(1)

Corrosion Cast Replication Procedure

(1)

Specimen Mounting

(2)

Criteria for Selecting Specimen Adhesives

(2)

Specimen Coating for Conductivity

(3)

Sputter Coating Procedure

(1)

Estimation of Coating Thickness

(1)

Potential Problems Associated with Sputter Coating

(1)

Noncoating Techniques

(1)

Tannic Acid/Osmium Noncoating Technique

(1)

Specimen Storage

(1)

References

(2)

Ultramicrotomy

(44)

Shaping the Specimen Block

(6)

Rough Trimming by Hand

(1)

Shaping the Block Using Razor Blades

(1)

Rough Trimming by Machine

(3)

The Mesa Trimming Procedure

(1)

Thick Sectioning

(2)

Fine Trimming

(1)

Types of Ultramicrotome Knives

(8)

Glass Knives

(1)

Preparing Glass Knives with a Commercial Knifemaker

(1)

Manually Crafted Knives

(1)

Breaking Glass Knives by Hand

(1)

Evaluation of Glass Knives

(2)

Attachment of Water Trough or Boat

(1)

Diamond and Sapphire Knives

(1)

Cleaning Diamond Knives

(1)

Histo Knives

(1)

Trough Fluids

(1)

Grids and Specimen Supports

(6)

Grids

(2)

Handling Grids

(1)

Support Films for Grids

(1)

Plastic Films

(3)

Pure Carbon Films

(1)

The Ultramicrotome and the Sectioning Process

(11)

Development of the Ultramicrotome

(1)

Basic Features of All Ultramicrotomes

(1)

The Sectioning Process

(1)

General Steps in Ultramicrotomy

(2)

Water Level Adjustment

(1)

Clearance Angle Adjustment

(1)

Knife Advancement

(1)

Specimen Orientation

(1)

Knife Contact

(1)

Ultrathin Sectioning

(1)

Section Retrieval

(2)

Collecting Serial Sections onto Single Hole or Slot Grids

(1)

Factors Affecting Sectioning

(1)

Methodical Sectioning

(1)

A Guide to Sectioning Problems and Causes

(1)

Cryoultramicrotomy

(9)

Dry Knife, Dry Retrieval Method

(2)

Dry Knife, Wet Retrieval Method

100

(4)

Example of the Use of Frozen Sections in an Immunocytochemical Localization Procedure

104

(2)

References

106

(2)

Specimen Staining and Contrast Methods for Transmission Electron Microscopy

108

(26)

Positive Staining

110

(8)

The Physical Basis of Contrast

111

(1)

Preembedding, Positive Staining with Uranyl Salts

111

(1)

Preembedding Staining Procedure

111

(1)

Postembedding Staining with Uranyl Salts

112

(1)

Staining Trimmed Specimen Blocks

112

(1)

Staining Trimmed Specimen Blocks in Uranyl Acetate

112

(1)

Staining Ultrathin Sections with Uranyl Salts

112

(1)

Helpful Suggestions when Staining

113

(2)

Staining Methods for Alcoholic Solutions of Uranyl Salts

115

(1)

Postembedding Lead Staining

115

(1)

Reynolds' Lead Citrate

115

(1)

Preparation and Use of Reynolds' Lead Citrate Stain

115

(1)

Preparing CO2-Free Water

116

(1)

Other Methods for Lead Citrate Staining

117

(1)

Staining Many Grids

118

(1)

Negative Staining

118

(3)

Commonly Used Negative Stains

118

(1)

Preparation of Three Commonly Used Negative Stains

118

(2)

Negative Staining Procedures

120

(1)

Metal Shadowing Techniques

121

(11)

Metal Evaporation Procedures

121

(1)

Vacuum System

121

(1)

Methods for Metal and Carbon Evaporation

122

(3)

Some Applications of Metal Shadowing and Negative Staining

125

(1)

Making Height Measurements Using Metal Shadowing

125

(1)

Replication of Biological Surfaces for Transmission Electron Microscopy

126

(1)

Preparing Single-Stage Replicas

127

(1)

Preparing Two-Stage, Negative Replicas

128

(1)

Visualizing Macromolecules

129

(1)

DNA Spreading and Shadowing Technique

130

(2)

References

132

(2)

The Transmission Electron Microscope

134

(50)

Visible Light, Electrons, and Lenses

136

(7)

Electromagnetic Radiation and the Diffraction Phenomenon

136

(1)

Effect of Diffraction on Resolution

137

(2)

Electrons, Waves, and Resolution

139

(1)

General Design of Lenses

140

(1)

Design of Electromagnetic Lenses

141

(2)

Defects in Lenses

143

(3)

Magnification

146

(1)

Design of the Transmission Electron Microscope

147

(23)

Comparison of Light Microscope to Transmission Electron Microscope

147

(1)

Basic Systems Making Up a Transmission Electron Microscope

147

(1)

Illuminating System

147

(9)

Specimen Manipulation System

156

(2)

Imaging System

158

(5)

Vacuum System

163

(1)

Vacuum Terminology

163

(5)

Vacuum Problems and Safety Features

168

(1)

Other Types of Vacuum Pumps

169

(1)

Preparing the Transmission Electron Microscope for Use

170

(12)

Alignment Theory

170

(1)

Alignment Practice

171

(2)

Major Operational Modes of the Transmission Electron Microscope

173

(1)

High Contrast

173

(1)

How to Obtain High Contrast

173

(2)

High Resolution

175

(1)

Darkfield

176

(1)

Diffraction

176

(1)

Diffraction Practices

177

(1)

Checking Performance

177

(1)

Alignment

177

(1)

Electrical Stability

178

(1)

Image Drift

178

(1)

Contamination

178

(1)

Magnification

179

(1)

Magnification Calibration

179

(1)

Resolution

180

(1)

Levels of Usage of the Transmission Electron Microscope

181

(1)

Shared Facilities

182

(1)

References

182

(2)

The Scanning Electron Microscope

184

(30)

Basic Systems of the SEM

186

(13)

Electron Optical and Beam Management Systems

187

(2)

Condenser Lenses 1 and 2

189

(1)

Final Condenser Lens

189

(1)

Dual Magnification Mode

190

(2)

Apertures and Depth of Field

192

(2)

Interaction of Electron Beam with Specimen

194

(1)

Specimen Manipulation

195

(1)

Electron Detector, Signal Processing, and Recording Systems

195

(1)

Signal Versus Noise

195

(1)

The Origin of Noise in the SEM

195

(1)

Secondary Electron Detector

195

(1)

Signal Processing

196

(1)

Some Specialized Features of the SEM

196

(3)

Image Recording

199

(1)

Films for Use in the SEM

199

(1)

Some Useful Features in Modern SEMs

199

(1)

Three Dimensionality of the SEM Image

199

(2)

Stereo Imaging with the SEM

201

(3)

Generating Two Micrographs with Separate Views

201

(1)

Using Tilt to Generate Stereo Views in the SEM

202

(1)

Merging Two Micrographs with Separate Views to Generate a Stereo Image

202

(1)

Preparing Stereo Images for Projection

203

(1)

Major Operational Modes of the SEM

204

(1)

High Resolution

204

(1)

Conditions for High Resolution

204

(1)

Great Depth of Field

205

(1)

Imaging Other Types of Specimen Signals

205

(3)

Backscattered Electrons

205

(1)

Backscattered Electron Detection

205

(2)

Types of Backscattered Electron Detectors

207

(1)

Important Considerations when Using Backscattered Imaging with Biological Specimens

207

(1)

Cathodoluminescence

208

(1)

One Type of Cathodoluminescence Detector

208

(1)

Using Secondary and Backscattered Electrons in Biological Studies

208

(2)

Specialized Instrumentation for Observing Unfixed Tissues

210

(3)

Observation of Frozen Specimens

210

(1)

Observation of Fresh Specimens

211

(2)

References

213

(1)

Production of the Electron Micrograph

214

(20)

Photographic Principles

216

(6)

General Steps in Producing an Electron Micrograph

216

(1)

Negative Recording Medium

216

(1)

Exposure Process in the SEM

217

(1)

Exposure Process in the TEM

217

(1)

Grain Versus Noise

218

(1)

Speed and Resolution

218

(1)

Improving Resolution and Contrast in TEM Negatives

219

(1)

Commercial Films, Handling, Developing, and Troubleshooting

219

(1)

Negative Recording Media for TEM

219

(1)

TEM Negative Media

219

(1)

Handling of Negative Materials and Processing

220

(2)

Standardization of Procedures

222

(1)

Problems with Negatives

222

(1)

Darkroom Printing

222

(8)

Work Prints and Final Prints

223

(1)

The Enlarger and Accessories

224

(1)

Printing Papers

225

(1)

Enlarging

226

(1)

Print Processing

227

(2)

Burning-in and Dodging

229

(1)

Techniques to Enhance Contrast

229

(1)

Matte and Glossy Electron Micrographs

229

(1)

Preparing Micrographs for Publication

230

(3)

The Final Print

230

(1)

Trimming Prints

230

(1)

Mounting Prints

230

(1)

Labeling Prints

231

(1)

Reproducing Prints

231

(1)

Slide Presentations

232

(1)

Poster Presentations

232

(1)

Making Montages

232

(1)

Determining Print Magnification from a Negative

232

(1)

References

233

(1)

Immunocytochemistry

234

(18)

The Antigen-Antibody Reaction

236

(1)

Approaches to Labeling

237

(2)

Ultrastructural Tags

239

(4)

General Considerations in Performing an Immunocytochemical Experiment

243

(5)

Immunohistochemistry/Immunofluorescence

243

(1)

Obtaining and Applying the Primary Antibody

244

(1)

Obtaining the Secondary Antibody

245

(1)

Tissue Fixation

245

(1)

Preembedding or Postembedding Labeling

245

(1)

Embedding

246

(1)

Blocking

246

(2)

Staining

248

(1)

Controls

248

(1)

Adsorption

248

(1)

Use of Tag or Unlabeled Antibody

248

(1)

Omission of Primary or Secondary Antibodies

248

(1)

Use of Pre-Immune Sera

248

(1)

Dealing with Soluble Antigens

248

(1)

Multiple Labeling Option

249

(1)

Interpretation of Micrographs

249

(1)

References

250

(2)

Enzyme Cytochemistry

252

(10)

Basis of Enzyme Cytochemistry

254

(1)

Requirements for Performing Enzyme Cytochemistry

254

(1)

Preservation of Tissue Structure and Enzymatic Activity

255

(1)

Maximization of Reaction Conditions

255

(1)

Facilitation of Substrate Penetration

255

(1)

Use of Appropriate Controls

255

(1)

Visualization of Reaction Product

255

(1)

Trapping Agents

255

(1)

Marker Enzymes

256

(1)

A Typical Protocol

257

(1)

Examples of Cytochemistry for Selected Enzymes

258

(2)

References

260

(2)

Autoradiography/Radioautography

262

(16)

Radioactivity

265

(1)

Emulsion Used in Autoradiography

266

(1)

How to Perform Autoradiography

267

(4)

Planning the Experiment

268

(1)

The Experiment Protocol

269

(1)

Administration of the Radioactive Substance and Tissue Preparation

269

(1)

Light Microscope Autoradiography

269

(1)

Application of Emulsion

269

(1)

Exposure of the Autoradiograph

269

(1)

Development of the Autoradiograph

270

(1)

Staining of the Tissue

270

(1)

Placement of the Exposed Tissue on Grids

271

(1)

Interpretation of Autoradiographs

271

(4)

A Typical Experiment Employing Autoradiography

272

(3)

References

275

(3)

Miscellaneous Localization Techniques

278

(8)

Actin

280

(1)

Carbohydrates/Oligosaccharides

280

(1)

Using Lectins

280

(1)

Using Tannic Acid and Metals

280

(1)

Using a Modified PAS-Schiff Reaction

281

(1)

Using Colloidal Iron and Colloidal Thorium

281

(1)

Golgi Complex/Multivesicular Body

281

(1)

Glycogen/Membranes

281

(1)

Ions

281

(1)

Nucleic Acids (DNA/RNA)

281

(2)

Protein

283

(1)

Sterols

283

(2)

References

285

(1)

Quantitative Electron Microscopy

286

(20)

When to Use Stereology

289

(1)

General Scheme of Stereology

289

(1)

Parameters Measured and Symbols Used in Stereology

290

(1)

Tissue Compartments or Spaces

290

(1)

Test Systems

291

(1)

Basic Types of Determinations and Associated Formulas

292

(7)

Volume Determinations

292

(2)

Volume Determination Methods for Spherical Objects

294

(1)

Volume Determinations for Nonspherical Objects

294

(3)

Area Determinations

297

(1)

Surface Density and Surface-to-Volume Determinations

298

(1)

Numerical Density Determinations

299

(1)

Assumptions and Conditions

299

(1)

Multiple Stage Sampling

300

(1)

How Much Data from Test Systems is Needed?

301

(1)

Computer-Assisted Stereology

301

(2)

A Typical Published Report

301

(2)

References

303

(3)

Freeze Fracturing Replication

306

(24)

How to Produce a Replica

311

(2)

Interpretation of Freeze Fracture Replicas

313

(6)

Finding a Membrane

313

(1)

Orienting the Micrograph so that the Shading of IMPs is from Below

314

(3)

Determining Which Membrane is being Viewed

317

(1)

Determining Whether the Membrane Face being Viewed is a P-Face or an E-Face

317

(2)

Complementary Replicas

319

(1)

Freeze Etching

319

(1)

Quick Freeze, Deep Etch, Rotary Shadow Techniques

320

(1)

Freeze Fracture Cytochemistry

320

(3)

Problems and Artifacts

323

(5)

References

328

(2)

The Analytical Electron Microscope

330

(28)

Interaction of an Electron Beam with a Specimen

332

(1)

Microscopes Used for Detecting Analytical Signals

333

(1)

X-Ray Microanalysis

334

(6)

Continuum (Bremsstrahlung) X Rays

335

(1)

Characteristic X Rays

336

(1)

The Filling of Inner Orbital Electrons is an Orderly Process

336

(1)

X-Ray Microanalysis may be Conducted to Achieve Several Goals

337

(1)

Equipment for Detecting X Rays

337

(3)

Information Obtainable Using X-Ray Analysis

340

(1)

Specimen Preparation for X-Ray Microanalysis

340

(4)

Bulk Samples

340

(1)

The Challenge: Quantitative Analysis of Bulk Specimens

341

(1)

Single Cells, Isolated Organelles, Liquid Secretions or Extracts

342

(1)

Sectioned Materials

342

(1)

Some Precautions with Sectioned Materials for X-Ray Microanalysis

343

(1)

Electron Energy Loss Spectroscopy (EELS)

344

(3)

Electron Diffraction

347

(8)

Formation of Diffraction Patterns

348

(1)

Single Crystal Versus Polycrystalline Specimens

349

(1)

Determination of Spacings in a Crystalline Lattice

350

(1)

Determination of Camera Constant (λL)

351

(2)

Types of Diffraction Modes

353

(1)

Selected Area Diffraction (SAD)

353

(1)

Microdiffraction

354

(1)

Other Types of Diffraction

355

(1)

References

355

(3)

Intermediate and High Voltage Microscopy

358

(10)

Historical Perspective

360

(1)

Advantages of High Voltage Microscopy

360

(3)

Contributions of High Voltage Microscopy

363

(4)

References

367

(1)

Tracers

368

(8)

Some Specific Tracers in Use

371

(4)

Cationic and Native Ferritin

371

(1)

Lanthanum

372

(1)

Horseradish Perioxidase

373

(1)

Lactoperoxidase

373

(1)

Ruthenium

374

(1)

References

375

(1)

Interpretation of Micrographs

376

(30)

Introduction to Viewing Biological Electron Micrographs

378

(1)

Interpretation of Normal Tissue Structure

379

(22)

Magnification and Resolution

379

(1)

Membranes

379

(1)

Shape, Kinds, and Number of Structures

380

(6)

Fixation Artifacts

386

(4)

Dehydration, Infiltration, and Embedding Artifacts

390

(1)

Sectioning Artifacts

390

(4)

Staining Artifacts

394

(2)

Microscope Artifacts

396

(2)

Photographic Artifacts

398

(3)

Interpreting Dynamic Processes from Static Images

401

(1)

Estimation of Micrograph Magnification

401

(3)

References

404

(2)

Survey of Biological Ultrastructure

406

(90)

The Cell Surface

408

(18)

The Lipid Bilayer of the Plasmalemma

409

(1)

The Glycocalyx

410

(1)

Cell Junctions

411

(8)

Cell Surface Specializations

419

(7)

The Cytoskeleton

426

(4)

Microtubules

426

(2)

Microfilaments

428

(1)

Intermediate Filaments

429

(1)

The Nucleus

430

(11)

The Nuclear Envelope

431

(2)

Chromatin

433

(1)

The Nucleolus

434

(2)

Dividing Cells

436

(4)

The Synaptonemal Complex

440

(1)

Mitochondria

441

(5)

Protein Synthetic and Secretory Structures

446

(10)

Free Ribosomes

446

(1)

Membrane Bound Ribosomes

447

(1)

Rough Endoplasmic Reticulum

448

(2)

Smooth Endoplasmic Reticulum

450

(1)

The Golgi Apparatus

451

(3)

Secretory Products

454

(2)

Centrioles

456

(1)

Cilia and Flagella

457

(3)

Cilia

457

(1)

Flagella

457

(3)

The Lysosomal System

460

(7)

Lysosomes

460

(3)

Multivesicular Bodies

463

(4)

Microbodies

467

(1)

Annulate Lamellae

468

(1)

Cell Inclusions

469

(5)

Glycogen

469

(2)

Lipid

471

(2)

Crystalloids

473

(1)

Extracellular Material

474

(4)

Collagen

474

(1)

Basal Lamina

475

(1)

Matrix of Bone and Cartilage

476

(2)

Special Features of Plant Tissues

478

(3)

Chloroplasts

478

(1)

Vacuoles

479

(1)

The Cell Wall

480

(1)

Bacteria

481

(2)

Algae, Fungi, Yeast, and Protozoa

483

(4)

Viruses

487

(2)

References

489

(7)

Safety in the Electron Microscope Laboratory

496

(25)

Personal Safety in the Laboratory

498

(3)

Safety Apparatus and Safe Practices

498

(2)

Pathogens and Radioisotopes

500

(1)

Chemical Safety

501

(6)

Handling Chemicals in a Safe Manner

501

(1)

Storage of Chemicals

502

(1)

Storage Recommendations

502

(1)

Some Chemicals Commonly Used in Electron Microscopy

503

(1)

Disposal of Spent Chemicals

504

(1)

Safety Monitoring

505

(1)

Cleaning Up Hazardous Spills

505

(1)

Exposure to Chemicals

506

(1)

New OSHA Standard

507

(1)

Fire Safety

507

(2)

Preventing Fires

507

(1)

Stopping Fires

508

(1)

Classes of Fires and Proper Extinguishers

508

(1)

Plan of Action for Fire Fighting

509

(1)

Electrical Safety

509

(1)

Darkrooms

509

(1)

Vacuum Evaporators and Sputter Coaters

509

(1)

Proper Grounding of Equipment

509

(1)

Servicing of Electron Microscopes and Small Equipment

509

(1)

First Aid for Shock Victims

510

(1)

Physical and Mechanical Hazards

510

(5)

Sharp Objects

510

(1)

Critical Point Dryers (CPDs)

510

(1)

Vacuum Pumps

510

(1)

Vacuum Evaporators

511

(1)

Sputter Coaters

511

(1)

Ovens

511

(1)

Cryogenic Gases and Vacuum Dewars

511

(1)

Compressed Gas Safety

512

(2)

Microwave Ovens

514

(1)

Radiation

514

(1)

Centrifuges

514

(1)

Pipetting

515

(1)

Falls

515

(1)

Training and Orientation Programs

515

(1)

Hotlines and Other Resources

516

(2)

Telephone Numbers

516

(1)

Addresses

516

(2)

References

518

(3)

Appendix A Review Questions and Problems

521

(10)

Appendix B Standard Units of Measurement

531

(2)

Index

533

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Electron Microscopy : Principles and Techniques for Biologists

Summary

Table of Contents

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