Biological Electron Microscopy

Theory, Techniques, and Troubleshooting 2nd Edition

Biological Elecfron Microscopy Theory, Techniques, and Troubleshooting

2nd Edition

Michael J. Dykstra and laura E. Reuss North Caro/ina State University

SPRINGER SCIENCE+BUSINESS MEDIA, LLC

Library ofCongress Cataloging-in-Publication Data

Dykstra, Michael J. Biological electron microscopy : theory, techniques, and troubJeshooting / Michael 1.

Dykstra and Laura E. Reuss.-- 2nd ed. p. cm.

Includes bibliographical references and index. ISBN 978-1-4613-4856-6 ISBN 978-1-4419-9244-4 (eBook) DOI 10.1007/978-1-4419-9244-4 1. EJectron microscopy. 2. Scanning electron microscopy. 3. Transmission electron

microscopy. 1. Reuss, Laura E. II. Title.

QH212.E4D952003 570'.28'25--dc21

© 2003 Michael 1. Dykstra Originally published by Kluwer Academic PublisherslPlenum Publishers Softcover reprint of the hardcover 2nd edition 2003 hrtp:/lwww.wkap.n/

10 9 8 7 6 5 4 3 2 I

AII righls reserved.

2003047709

No part of Ihis work may be reproduced, slOred in a retrieval syslem, or transmined in any form or by any means, electronic, mechanical, pholOcopying, microfilming, recording, or olherwise, wilhoul wrillen permission from Ihe Publisher, wilh Ihe exception of any material supplied specifically for the purpose of being entered and execuled on a computer syslem, for exclusive use by the purchaser of Ihe work.

ISBN 978-1-4613-4856-6

Preface

Electron microscopy is frequently portrayed as a discipline that stands alone, separated from molecular biology, light microscopy, physiology, and biochemistry, among other disciplines. It is also presented as a technically demanding discipline operating largely in the sphere of "black boxes" and governed by many absolute laws of procedure. At the introductory level, this portrayal does the discipline and the student a disservice. The instrumentation we use is complex, but ultimately understandable and, more importantly, repairable. The procedures we employ for preparing tissues and cells are not totally understood, but enough information is available to allow investigators to make reasonable choices concerning the best techniques to apply to their parti­cular problems. There are countless specialized techniques in the field of electron and light microscopy that require the acquisition of specialized knowledge, particularly for interpretation of results (electron tomography and energy dispersive spectroscopy immediately come to mind), but most laboratories possessing the equipment to effect these approaches have specialists to help the casual user. The advent of computer operated electron microscopes has also broadened access to these instruments, allowing users with little technical knowledge about electron microscope design to quickly become operators. This has been a welcome advance, because earlier instru­ments required a level of knowledge about electron optics and vacuum systems to produce optimal photographs and to avoid "crashing" the instruments that typically made it difficult for beginners.

There are many books and book series that deal with biological electron microscopy, but there are only few individual texts that give a comprehensive overview of preparative techniques and instrumentation that can answer the myriad questions posed by those pursuing structure­function relationships of cellular materials. Many students are taught to fear, rather than respect, electron microscopy instrumentation. In addition, many texts continue to teach that there is only one right way to fix and embed a given class of organisms, only one way to properly break a glass knife, only one side of a grid to use for section retrieval, and only one way to properly post-stain grids. After spending over 30 years reading about all the different types of approaches utilized to obtain publishable ultrastructural work, it is obvious that there are numerous equally valid methods to approach various questions, all of which will produce publishable results.

This textbook is an updated blend of technical approaches and didactic information found in the two books, Biological Electron Microscopy: Theory, Techniques, and Troubleshooting (Dykstra, 1992) and A Manual of Applied Techniques for Biological Electron Microscopy (Dykstra, 1993). This book is intended for a one-semester course that covers all the basic approaches to light microscopy, transmission electron microscopy, and scanning electron microscopy. Sections have been added to address photomicroscopy (including confocal scanning microscopy), digital imaging, and electron tomography utilizing intermediate voltage transmission electron microscopes.

A major component of this book is suggested by the subtitle: Theory, Techniques, and Troubleshooting. Too many electron microscopists have been trained with little theory beyond optical theory, too little about techniques except the ones used in their specific laboratory, and almost nothing about troubleshooting problems, particularly with regard to instrumentation. In a discipline with so many varied approaches from which to choose, learning how to apply

v

vi Preface

appropriate preparative techniques and choosing instrumentation for approaching questions con­cerning cell biology logically probably represent the highest aim of a course in electron microscopy.

This text is definitely not an in-depth compendium of all of the techniques and instrumen­tation capabilities currently available. Such an endeavor would occupy countless volumes (as exemplified by the excellent series edited by Audrey Glauert, Practical Methods in Electron Microscopy). What we have tried to accomplish is to put forth some basic tested techniques for common needs in the Techniques section at the end of various chapters, along with basic expla·· nations of the different technical approaches and instrumentation employed, so that a student can see what is possible and can see what methods can be used to answer the variety of questions posed by cell biology in the cytological arena. To do this in one text suitable for a one-semester course necessitates brevity and superficiality in some areas, but the literature citations are intended to allow students to take their quest for knowledge in a specific area to a higher level.

A student who masters the concepts in this text will be capable, with continued practice on technical skills, to productively utilize electron and light microscopy techniques in his or her research. We hope that this text will also provide a sufficient foundation from which students can expand their horizons to numerous other specific areas of expertise within biological research.

Michael 1. Dykstra January 19,2003

Acknowledgments

When the first edition of this book was written, acknowledgments were not included, which was an oversight corrected when A Manual of Applied Techniques for Biological Electron Microscopy (Dykstra, 1993) was published. Since the current textbook is a compilation of the materials found in both of these books, it seems appropriate to recap the acknowledgments from the manual and to include the acknowledgments that should have been in the first edition of this book.

Thus, this manual is dedicated to all of our friends, colleagues, technicians, and students who have helped us develop these approaches and have tested the various specific recipes and pro­cedures to demonstrate that they generate reproducible results. We have been fortunate to have had a number of excellent technicians who have generated the bulk of the work coming out of our current laboratory (the Laboratory of Advanced Electron and Light Optical Methods, or LAELOM) and other laboratories which we have overseen over the years. We are particularly indebted to the following individuals who have made laboratory life instructive and easier than it would have been otherwise:

Sarah Bierley Brendalyn Bradley-Kerr Karen Greer Jacqueline Lee Nina Rodenroth Robert Seiler

None of this work could have been accomplished without the formative 2 years that the senior author (Michael J. Dykstra) spent working as a post-doc for Dr. Henry C. Aldrich at the University of Florida. He introduced the concept that electron microscopes are not holy temples, but are just another type of machine that can be tinkered with, adjusted, and fixed. He also pro­vided the concept that understanding the mechanism of fixation protocols and other methods makes it easier to make cogent choices when working with a new system or group of organisms. After two years with Dr. Aldrich, electron microscopy made much more sense and the intimidat­ing aspects of earlier training were erased, which has made the ensuing 25 years in the field of electron and light microscopy much easier and more productive than it would have been otherwise.

Finally, Mary Born, the editor of the first two books that have been blended in this text, convinced the senior author to begin the daunting task of writing textbooks and helped him learn how to write more clearly. Without her encouragement, corrections, and helpful suggestions, the original books would not have come to be. Authors, in general, could not be what they are with­out the help of thoughtful editors. We were fortunate to walk the path that led to this book guided by such an inspiring soul.

vii

Introduction to the Second Edition

Since the first edition of this book appeared in 1992, there has been an incremental improvement in most of the instrumentation utilized by electron microscopists. Conventional transmission electron microscopes (TEMs) operating in the 80-120kV range and conventional scanning electron microscopes (SEMs) have generally been adapted to run in a Microsoft Windows™ environment. Specialized instrumentation, including intermediate voltage electron microscopes (IVEMs), high­resolution scanning electron microscopes, and environmental scanning electron microscopes (ESEMs) have become easier to use, more reliable, and are more commonly found on university cam­puses than in previous years. Advances in digital imaging and electron tomography techniques have changed our methods of image acquisition and storage and improved our understanding of complex three-dimensional structures. Finally, incremental advances in microanalytical packages (both in hard­ware and software), ultrarnicrotomes, cryoultramicrotomes, and various devices involved in preparing or viewing cryosamples have increased the ease of access to these techniques to biological researchers.

The transition from single department research electron microscopy facilities to multi-user service facilities serving campus-wide constituencies has been necessitated by the ever-increasing cost of instrumentation and maintenance. A fortuitous outcome of this shift in resource placement has been that the remaining facilities have greater pooled resources, allowing the purchase of more expensive and more broadly capable instrumentation in many cases.

Specimen preparation techniques have not changed much since the last edition of this book. The epoxide resins, acrylic resins, and fixatives used today were all available in 1992. The equipment for processing samples, both for conventional resin embedding and for cryotechniques, have improved, but no major breakthroughs have occurred. For this reason, the sections dealing with these subjects will be quite familiar to readers of the previous edition. The addition of a spe­cific methods section at the end of selected chapters, however, is a major change from the first edi­tion and we hope that this will help the reader to quickly jump from the didactic material to the direct application of techniques to their research problems.

Electron microscopy societies have, in general, broadened their bases by adding light microscopy approaches to their traditional interest in electron microscopy methodologies. In par­ticular, the rapid rise in the importance of confocal microscopy to cytological investigations has filled in some visual gaps between light microscopy and electron microscopy, creating a fairly seamless path from images produced by compound light microscopes through those from confo­cal microscopes to those generated by transmission and scanning electron microscopes. To reflect this broadening, for example, the Electron Microscopy Society of America (EMSA) became the Microscopy Society of America (MSA). These changes seem fitting, particularly since electron microscopists have always used light microscopy methods during their investigations.

With that said, we asked ourselves why a new edition of our book(s) seemed sensible. As mentioned above, we decided that a new edition would allow us to merge the two original texts so that the didactic survey of different approaches to instrumentation and techniques could be immediately followed by specific techniques that we felt we could recommend, based on our years using them in our service laboratory. Having all of this material in one place, rather than in two separate volumes, was seen as a better package for the reader.

ix

x Introduction to the Second Edition

Some of the newer instruments gaining prominence in the 1990s such as IVEMs, low vacuum scanning electron microscopes (LV SEMs), FEG-equipped SEMs and TEMs, and ESEMs are now widely available and have increased the variety of capabilities for cytological obser­vations considerably. The most notable advance has been the development of the discipline of electron tomography, which has produced scores of papers in the last 10 years and has allowed the characterization of the structure of macromolecules and cellular organelles as well as elucidating the three-dimensional relationships between cellular constituents with an ease and clarity only dreamed about by most electron microscopists in the 1980s.

The digital revolution that is affecting all aspects of visual communication has led to the digital control of microscopes, production of digital images, and the concomitant problems asso­ciated with producing appropriate digital files for their intended purposes, both from electron microscopes and from light microscopes. As you will see, we still believe film-based photography has a place with monochromatic images, though color digital images are a more pragmatic approach to producing printable color images.

We hope that the discussions of instrumentation and technical approaches available for cytological investigations will stimulate the reader to embark on the exciting adventure of study­ing cells and tissues. We are devoted to the concept that understanding structure/function rela­tionships between what is in a cell and what the cell is doing are invaluable. We also encourage all students of cytological approaches to recognize that research should not be designed to use tools. Instead, tools should be chosen on the basis of the scientific questions being asked. Cytological, physiological, biochemical, and molecular tools should be used in concert, at a min­imum, to study cellular behavior. Thus, it is appropriate for biologists utilizing electron microscopy to describe themselves from the standpoint of their biological discipline (e.g., botany, entomology, microbiology), rather than to describe themselves as electron microscopists. The physicists and electronics engineers, starting with Ernst Ruska, who have made possible the stunning array of electron microscopes available today are the true electron microscopists.

The purpose of this text is to help students see what is possible and to help them approach instrumentation and preparative techniques in a thoughtful, scientific way, so that they can solve microscopy and sample handling problems quickly and easily so that they can get on with their investigations of cells and tissues.

When exploring the technical approaches we recommend, we hope you realize that we are not trying to be encyclopedic. The techniques described are included because we routinely use them with great success. They will not fulfill every need for every sample, but they work for the vast majority of samples and they should serve as an excellent starting point, if no other spe­cific approach has been gleaned from the literature pertaining to your specific biological model.

We hope that the journey through the myriad types of instrumentation and associated sam­ple preparation procedures will serve to introduce you to a set of disciplines associated with cyto­logical investigations that have kept the senior author fascinated with cellular structure and function for over 30 years as he has used electron microscopy to study aspects of protozoan and fungal development in his own research. Over the years, studies of bacteria, viruses, mammalian tissues, insects, fish, reptiles, amphibians, nematodes, and various types of cytopatholgy were added to the mix. In recent years, our laboratory has devoted a major amount of effort to the assessment of changes induced in animal tissues produced during drug development studies. There seems to be an infinite variety of biological questions that can be answered, at least in part, by cytological approaches.

We hope that the introduction to this discipline that is in your hands will lead you to cyto­logical research and that it will give you as much thoughtful stimulation and entertainment as we have experienced during our years as biologists using electron and light microscopy.

Contents

Chapter 1 Specimen Preparation for Electron Microscopy

I. Physical Fixation Techniques II. Traditional Chemical Fixation

III. Buffers IV. Dehydration V. Embedding Media

VI. Examination of Tissues Prepared with a Variety of Fixatives and Buffers VII. A Quasi-Universal Fixation, Dehydration, and Embedment Schedule

Successfully Used for Organisms from the Five Major Kingdoms of Life References

Chapter 1 Techniques

Making Dilutions Diluting Stock Solutions Preparing Stock Solutions to Be Mixed Together to Achieve Specific Final

Concentrations in a Working Fixative Solution A Routine Fixation and Embedding Schedule for Transmission Electron

Microscopy Samples (Tissues or Cells) Preparation of Primary (Aldehyde) Fixatives Preparation of Osmium (Osmium Tetroxide, Osmium Tetraoxide) Buffering Systems

Cacodylate Buffers Phosphate Buffers Tris-HCl Buffer Sodium Acetate Buffer Dulbecco's Phosphate-Buffered Saline

Resin Formulations Spurr Resin PolylBed 812 Resin SPI-Pon 812 Araldite 6005 Resin Mollenhauer's EponiAraldite Resin (Adapted for Use with Epon Substitutes) London Resin Co. (LR) White Resin Lowicryl and LR Gold Resins

PEG Method for TEM Sections JB-4™ (Glycol Methacrylate) Techniques for High-Resolution Light Microscopy Agar Embedment of Cell Suspensions or Subcellular Particulates for TEM Preparing Adherent Tissue Culture Monolayers in Situ for TEM

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1

1 2

24 29 31 36

40 72

74

74 74

74

74 80 81 85 86 87 89 90 91 92 93 96 97 99

100 101 103 104 105 107 109

XII

Flat Embedding of Cell Cultures Grown on Permanox® Tissue Culture Dishes for TEM

Preparation of Buffy Coats for TEM Sperm Fixation Central Nervous System Fixation (Brain, Spinal Cord) Using Vacuum to Help Wet Fungal, Plant, or Insect Samples during

Primary Fixation Simultaneous Glutaraldehyde/Osmium Fixation for Protozoan Samples or

Samples with a Large Amount of Lipid Killing Cells Prior to Chemical Fixation Flat Embedding on Microscope Slides Procedure for Deparaffining Samples

Chapter 2 Cryotechniques

I. History II. Purpose

III. Cryogens IV. Safety Precautions V. Freezing Methods

VI. Uses of Frozen Specimens VII. Artifacts and Their Correction References

Chapter 2 Techniques

Cryoultramicrotomy for Structural Examinations or Consequent Immunolabeling

Chapter 3 Ultramicrotomy

I. Ultramicrotomes II. Knives

III. Block Trimming IV. Ultrarnicrotomy Working Area

References

Chapter 3 Techniques

Making a Section Retrieval Loop Making a Section Manipulation Tool Making a Locking Ring for Forceps Making Glass Knives Making Glass Knife Boats Glass Knife Storage Block Trimming Sectioning Procedures Semi thin Sections Grid Selection

Contents

III 111 114 116

117

118 119 120 122

125

126 127 128 129 129 135 146 149

150

150

153

153 155 157 158 158

159

159 160 161 161 164 164 165 167 170 171

Contents

Grid Cleaning Ultrathin Sections Common Sectioning Problems

Chapter 4 Staining Methods for Semithins and Ultra thins

I. Semithin Section Staining II. Ultrathin Section Staining

References

Chapter 4 Techniques

Semithin Section Staining with Toluidine Blue 0 Polychrome Stain for Semithin Sections Staining Ultrathin Sections Subbing Slides

Chapter 5 Cytochemistry

I. Problems II. Specific Reaction Products

III. Examples of Enzyme Cytochemistry IV. Examples of Nonenzymatic Cytochemistry References

Chapter 5 Techniques

Polysaccharide Stains Ruthenium Red Staining Silver Methenamine Staining for Polysaccharides

Calcium Staining Prefixation Calcium Staining for Muscle Tissue Postfixation Calcium Staining with Pyroantimonate

Chapter 6 Immunocytochemistry

I. Purpose II. Preparative Techniques

III. Immunoglobulins IV. Common Immunolabeling Techniques for Electron Microscopy References

Chapter 6 Techniques

Colloidal Gold Techniques Preparation of 13-nm Colloidal Gold

xiii

171 172 172

175

175 179 189

190

190 191 192 196

197

197 199 200 203 207

209

209 209 210 213 213 215

219

219 220 224 226 230

232

232 233

XIV

Conjugation of Gold to Proteins Indirect Immunolabeling Procedure for Sections of Materials

Embedded in LR White or Lowicryl K4M Resin (Acrylic Resins) Procedure for Immunolabeling Intact Cells

(Preembedment Labeling of Cell Surfaces)

Chapter 7 Support Films

1. Purpose II. Types

III. Methods References

Chapter 7 Techniques

Preparation of Formvar-Coated Grids Formvar-Coated Aluminum Bridges for Slot Grids Coating Grids with Butvar B-98 Coating Grids with Collodion Films Making Carbon Support Films

Chapter 8 Replicas, Shadowing, and Negative Staining

I. Shadow Casting II. Negative Staining

References

Chapter 8 Techniques

Vacuum Evaporation Shadow Casting DNA (Plasmid) Preparation for TEM

Negative Staining Negative Staining with Phosphotungstic Acid Negative Staining with Uranyl Acetate Negative Staining with Ammonium Molybdate Wetting Agents Used for Negative Staining Preparation of Virus Samples for Transmission Electron Microscopy Ultracentrifugation Technique for Viral Sample Preparation Immune Electron Microscopy for Concentrating Viruses Viral Concentration with the Beckman Airfuge

Chapter 9 Transmission Electron Microscopy

I. Historical Review of Microscopy (1590-2003) II. Theory of Electron Optics

Contents

235

237

238

241

241 241 242 244

245

245 250 252 253 256

259

259 266 269

271

271 271 274 275 276 279 280 280 281 282 283 284

287

287 288

Contents xv

III. Four Aspects of Image Formation 300 IV. General TEM Features 30 I V. Parts of the Electron Microscope: Functional Aspects 302

VI. Operation of the TEM: Decision Making 313 References 321

Chapter 10 Vacuum Systems 323

I. Types of Gauges 324 II. Vacuum Pumps 326

III. Sequential Operation of a Complete Vacuum System to Achieve High Vacuum 334 IV. Lubrication of Vacuum Seals and Leak Detection 336

References 337

Chapter 11 High-Voltage Transmission Electron Microscopes (HVEM) 339

I. History 339 II. Purpose 339

III. Functional Aspects of HVEMs 340 IV. Microscope Construction 342 V. Sample Preparation 344

VI. Applications 344 References 345

Chapter 12 Intermediate Voltage Electron Microscopes (IVEM), Electron Tomography, and Single-Particle Electron Microscopy 347

I. Intermediate Voltage Electron Microscopes 347 II. Electron Tomography and Single-Particle Electron Microscopy 349

References 355

Chapter 13 Scanning Electron Microscopy 357

I. History 357 II. The Use of SEM in Biological Research and Medicine 358

III. Principles of the SEM 360 IV. Operation of the SEM 362 V. Interaction of the Electron Beam and Specimen 363

VI. Specimen Preparation 371 VII. Artifacts and their Correction 376

VIII. Specialty SEMs: FEG, LV, and ESEM Instruments 377 References 382

Chapter 13 Techniques 384

PolY-L-Lysine Technique for Attaching Particulates to Coverglasses for SEM 384

XVI

Organosilane Coverglass/Slide Treatment Critical Point Drying for SEM Drying Samples with Hexamethyldisilazane Sputter Coating Vascular Casting with Mercox CL-2BTM Resin

Chapter 14 Microanalysis

I. Energy Dispersive Spectroscopy (EDS) II. Electron Energy Loss Spectroscopy (EELS)

References

Chapter 15 Photography

I. Emulsion Composition II. Film Types

III. Producing A Latent Image IV. Film Processing V. Development Controls

VI. Paper Types VII. Keeping Properties of Chemicals and Precautions

VIII. Sharpness IX. Films Commonly Used in the EM Laboratory X. Copy Work

XI. Types of Enlargers XII. Viewing a Print in Perspective

References

Chapter 15 Techniques

Kodak Electron Microscope (TEM) Film 4489 Films in 35-mm Format

Kodak Technical Pan 2415 Film for Photomicrography Ilford Pan-F and FP-4 Kodak T-Max 100 Film Kodak Kodalith Film Kodak LPD4/Precision Line Film Kodak Rapid Process Copy 2064 Film Tungsten-Balanced Kodak Ektachrome

Polaroid Copy Negatives Using Type 55 PIN Film Making Photographic Prints Poster Preparation

Chapter 16 Digital Imaging and Telemedicine

I. General Concepts Concerning Digital Imaging

Contents

385 386 388 389 391

395

395 401 402

405

406 407 409 409 412 413 414 415 415 417 422 424 425

426

426 427 427 428 429 430 431 431 432 433 434 437

439

439

Contents

II. Telemedicine/Telepathology Considerations References

Chapter 17 Morphometry and Stereology

I. Purpose II. Resolution and Discrimination

III. Image Processing IV. Stereology V. Computer-Assisted Analysis of Movement

References

Chapter 18 Photomicroscopy

I. Light-Microscope Objective Lenses II. Light-Microscope Oculars

III. Light-Microscope Condenser Assemblies IV. Slide Thickness V. Light Sources

VI. Types of Optical Systems References

Chapter 18 Techniques

Kohler Illumination Use of the Substage Condenser Diaphragm Focusing Using a Focusing Telescope (Bertrand Lens) Using a Stage Micrometer and Ocular Scale to Measure Objects or to

Calibrate Microscopes and Morphometry Programs Reading an Objective Lens Use of Focusing Collars on High Dry (40 X) Objective Lenses Using Oil- or Water-Immersion Objectives Use of Filters with Black-and-White Films Use of Filters with Color Films

Chapter 19 Laboratory Safety

Chapter 20 General Sources for Information Concerning Microscopy

I. Atlases II. Journals

III. Societies IV. National Resources (Institutional Instrumentation)

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446 449

451

451 452 452 455 457 458

459

459 465 466 467 467 468 479

480

480 480 481

481 482 483 484 485 486

489

491

491 491 492 492

xviii

Chapter 21 Electron Microscopy Equipment and Supplies

I. Expendable Supplies and Small Equipment II. Light Microscopes III. Electron Microscopes IV. Diamond Knives V. High-Vacuum Pumps

VI. Ultramicrotomes VII. Equipment for Cryotechniques

VIII. Sputter Coaters and Vacuum Evaporators

Appendix A Computing Micrometer Bar Sizes

Appendix B Calibrating the TEM and the SEM

I. Transmission Electron Microscope II. Scanning Electron Microscope

Appendix C Materials and Methods Write-Up Suggestions for Standard TEM and SEM Preparations

I. Materials and Methods for Routine TEM Preparation II. Materials and Methods for Routine SEM Preparation

Index

Contents

493

493 494 495 495 496 496 497 498

499

501

501 501

503 503 504

505