Biological Electron Microscopy. Theory, Techniques, and Troubleshooting

Transcription

1 Biological Electron Microscopy Theory, Techniques, and Troubleshooting

2 Biological Electron Microscopy Theory, Techniques, and Troubleshooting MICHAEL J. DYKSTRA College of Veterinary Medicine North Carolina State University Raleigh, North Carolina PLENUM PRESS NEW YORK AND LONDON

3 Library of Congress Cataloging in Publication Data Dykstra, Michael J. Biological electron microscopy: theory, techniques, and troubleshooting / Michael J. Dykstra. p. cm. Includes bibliographical references (p. ) and index. ISBN-13: e-isbn-13: DOl: / Electron microscopy. 2. Scanning electron microscopy. 3. Transmission electron microscopes. I. Title. QH212.E4D '.45-dc20 CIP ISBN-13: Michael J. Dykstra Softcover reprint ofthe hardcover 1st edition 1992 Plenum Press is a division of Plenum Publishing Corporation 233 Spring Street, New York, N.Y All rights reserved No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from the Publisher

4 To the two Susans, for patience

5 Preface Electron microscopy is frequently portrayed 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 description does the discipline and the student a disservice. The instrumentation we use is complex but ultimately understandable and, more important, reparable. The chemical and physical processes underlying the procedures we employ for preparing tissues and cells are not totally elucidated, 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 microscopy that require the acquisition of specialized knowledge, particularly for the interpretation of results (energydispersive spectroscopy comes immediately to mind), but most laboratories with equipment to utilize these approaches have specialists to help the casual user. There are several books available that deal with biological electron microscopy. Some (e.g., Meek, 1976) are extremely good in regard to instrumentation, whereas others are heavily skewed toward specimen preparation (e.g., Hayat, 1989). Some, such as that by Wischnitzer (1981), cover both specimen preparation and instrumentation in almost sufficient detail for an introductory course. Unfortunately, most singlevolume electron microscopy texts were written prior to 1983 and are thus quite dated. There are also several series of books (see the Hayat series and the Glauert series in Appendix B) containing quite complete and up-to-date information on many subjects, but these are too expensive and too extensive to be considered as textbooks. There are also a number of laboratory procedure manuals available, but these consistently state that the limited techniques they list are the only ones that can be used. This is clearly not the case. Most of my contemporaries were taught fear, rather than respect, for the instrumentation of electron microscopy. In addition, we were generally taught that there was 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 vii

6 viii PREFACE only one way to properly post-stain grids. After going to various meetings for 20 years and hearing all the different types of approaches utilized to obtain publishable ultrastructural work, I see clearly that the system is much more flexible than our training had suggested. This textbook is offered to help a beginning student see how things work in electron microscopy. It presents the principles of fixation and specimen preparation, showing the breadth of approaches that are practical. It gives students sufficient information about basic specimen preparation to make cogent decisions for their own projects after consulting the relevant literature for their own group of organisms or tissues. There are still black boxes in the discipline, but I will try to minimize them. A major component of this book is suggested by the subtitle: Theory, Techniques, and Troubleshooting. Too many of my generation were given in their training little theory beyond optical theory; too little information about techniques, except the ones used in their specific laboratory; and almost nothing about troubleshooting problems, particularly in regard to instrumentation. Clearly, things do go wrong, and with alarming regularity. In a discipline with so many varied approaches from which to choose, teaching how to approach a problem logically and how to correct a problem that presents itself probably represents the highest aim of a course in electron microscopy. This book is intended for a one-semester course that covers all the basic approaches utilized in transmission and scanning electron microscopy. Its arrangement is somewhat unusual in being more or less chronological, so that the student can be reading about the techniques as they are employed in class. Finally, one of the major purposes of this text is to provide more up-to-date coverage of the two areas that have revolutionized electron microscopy over the last 8-10 years: cryotechniques and immunolabeling. When glutaraldehyde was introduced by Sabatini et al. in 1963, chemical fixation more or less reached its apex. Until the advent of easily reproducible cryotechniques, no further advances took place in specimen preparation for transmission electron microscopy. By the late 1970s, the discipline was perceived by many as a fairly passe descriptive science with little innovation except in the area of high-voltage electron microscopy. Fortunately, in the 1980s there was improved instrumentation for cryo work, and the coupling of immunology and electron microscopy through immunogold and immunoperoxidase techniques has brought our discipline back on line. The power of current techniques to utilize molecular approaches to probe the location of various cellular products and activities has rekindled interest in the truly dynamic aspects of cellular behavior. A student who masters the concepts in this text will be capable, with continued practice in technical skills, to utilize electron microscopy techniques productively in his or her research. It is my hope that this text will give a firm foundation on which the student can build knowledge of cellular structure and behavior far beyond the scope of this work. Hayat. M.A Fixation for electron microscopy. Academic Press. New York. Meek, G.A Practical electron microscopy for biologists, 2nd ed. John Wiley & Sons, New York. Wi schnitzer, S Introduction to electron microscopy, 3rd ed. Pergamon Press, New York.

7 Contents Introduction 1 Chapter 1 Specimen Preparation for Transmission Electron Microscopy I. Fixatives... 5 A. Purpose; Killing versus Fixing... 5 B. Methods II. Buffers A. Molarity/Molality/Osmolarity/Tonicity B. Purpose C. Types, Characteristics, and Uses of Buffers III. Dehydration A. Purpose B. Agents IV. Embedding Media A. Ideal Qualities B. Classes and Characteristics of Resins C. Embedding Mold Types D. Agar Embedment V. Examination of Four Tissues Prepared with a Variety of Fixatives and Buffers VI. A Quasi-Universal Fixation, Dehydration, and Embedment Schedule Successfully Used on Organisms from All Five Kingdoms of Life.. 68 References Chapter 2 Ultramicrotomy I. Ultramicrotomes ix

8 x CONTENTS A. Purpose B. Design C. History II. Knives A. History B. Glass Knive Manufacturing C. Section Handling D. Knife Storage E. Diamond Knives III. Block Trimming A. Trimming Procedures B. Block-Trimming Tools IV. Sectioning Procedures A. Working Area B. Sectioning Procedures v. Eight Commonly Encountered Sectioning Problems References... ' Chapter 3 Support Films 103 I. Purpose II. Types A. Nitrocellulose B. Formvar C. Carbon III. Methods A. Droplet B. Slide Stripping C. Holey Films D. Carbon Films References Chapter 4 Transmission Electron Microscopy I. Historical Review of Microscopy ( s) II. Theory of Electron Optics A. Light Microscopes versus the TEM B. Resolution C. Electron Lenses D. Properties of Electron Lenses

9 CONTENTS xi III. Four Aspects of Image Formation A. Absorption B. Interference C. Diffraction D. Scattering IV. General TEM Features A. Operating Voltage B. Resolution C. Magnification D. High Vacuum, Electronic, Magnetic, and Physical Stability V. Parts of the Electron Microscope: Functional Aspects A. The Electron Gun B. Condenser Lens System C. Deflector Coils D. Objective Lens E. Diffraction Lens F. Projector System G. Camera Systems H. Specimen Holders I. Viewing System Detectors VI. Operation of the TEM: Decision Making A. Accelerating Voltage B. Choice of Beam Current and Bias C. Condenser Settings D. Objective Settings E. Alignment F. Taking a Photograph G. Specimen Radiation Dose H. Microscope Calibration References Chapter 5 Vacuum Systems 151 I. Types of Gauges A. Direct Reading B. Indirect Gauges II. Vacuum Pumps A. Types of Pumping Systems B. Mechanical/Rotary Vane Pumps C. Diffusion Pumps D. Turbomolecular Pumps

10 xii CONTENTS E. Sputter Ion (Ion Getter) Pumps F. Cryopumps and "Cold Fingers" III. Sequential Operation of a Complete Vacuum System to Achieve High Vacuum IV. Lubrication of Vacuum Seals and Leak Detection References Chapter 6 Staining Methods for Semithins and Ultrathins 167 I. Semithin Section Staining A. Toluidine Blue-O B. Toluidine Blue-O and Acid Fuchsin C. Basic Fuchsin/Methylene Blue D. Methylene Blue E. Periodic Acid/Schiff's Reagent II. Ultrathin Section Staining A. Purpose B. En Bloc versus Post-Staining C. Commonly Used Post-Stains D. Microwave Staining E. Dark-Field Imaging without Staining References Chapter 7 Photography I. Emulsion Composition II. Film Types III. Producing a Latent Image IV. Film Processing A. Developer B. Stop Baths C. Fixer V. Development Controls A. Time B. Temperature C. Agitation D. Developer Choice VI. Paper Types VII. Keeping Properties of Chemicals and Precautions VIII. Sharpness

11 CONTENTS xiii IX. Films Used in the Electron Microscopy Laboratory A. Negative-Release Films B. Positive-Release Films X. Copy Work A. Films B. Improving Copy-Stand Images XI. Types of Enlargers A. Diffusion Enlargers B. Condenser Enlargers C. Point Light Source Enlargers D. LogEtronics Enlargers XII. Viewing a Print in Perspective References Chapter 8 Replicas, Shadowing, and Negative Staining I. Shadowing Casting A. Mechanism B. Metals Used C. Vacuum Evaporators D. Electrodes E. Factors Leading to Fine Grains of Shadowed Metal F. Shadowing Techniques G. Sputter Coating II. Negative Staining A. Mechanism B. Methods References Chapter 9 Scanning Electron Microscopy 223 I. History II. The Use of SEM in Biological Research and Medicine III. Principles of the SEM IV. Operation of the SEM V. Interaction of the Electron Beam and Specimen A. Resolution B. Secondary Electrons C. Backscattered Electrons D. Auger Electrons

12 xiv CONTENTS E. Energy-Dispersive Spectroscopy (EDS) F. Cathodoluminescence VI. Specimen Preparation A. Fixation B. Dehydration and Transition Fluids C. Drying D. Mounting Specimens E. Coating Specimens VII. Artifacts and Their Correction VIII. Specialty SEMS: FEG, LV, and ESEM Devices References Chapter 10 Cryotechniques 247 I. History A. Organismal Period B. Mechanistic Period C. Cytological Period II. Purpose III. Cryogens IV. Safety Precautions V. Freezing Methods A. High-Speed Plunging/Immersion B. Spray Freezing C. Jet Freezing D. High-Pressure Freezing E. Metal Mirror/Slam Freezing VI. Uses of Frozen Specimens A. Cryoultramicrotomy B. Cryosubstitution C. Freeze-Fracture D. Freeze-Drying as Typified by Molecular Distillation VII. Artifacts and Their Correction References Chapter 11 High-Voltage Electron Microscopy 275 I. History II. Purpose III. Functional Aspects of HVEMS A. Resolution

13 CONTENTS xv B. Radiation Damage C. Contrast IV. Microscope Construction V. Sample Preparation VI. Applications VII. Intermediate-Voltage Electron Microscopy (IVEM) References Chapter 12 Microanalysis Microanalysis Techniques A. Energy-Dispersive Spectroscopy (EDS) B. Electron Energy Loss Spectroscopy (EELS) References Chapter 13 Cytochemistry 295 I. Problems II. Specific Reaction Products A. Peroxidase Procedures B. Lead Capture C. Ferritin D. Colloidal Gold E. Ruthenium Red, Alcian Blue, Pyroantimonate III. Examples of Enzyme Cytochemistry A. Peroxidase Methods B. Hatchett's Brown Methods C. Lead-Capture Methods IV. Examples of Nonenzymatic Cytochemistry A. Cationic Dyes B. Polysaccharide Stains C. Monosaccharide and Disaccharide Stains D. Calcium Staining References Chapter 14 Immunocytochemistry 309 I. Purpose II. Preparative Techniques

14 xvi CONTENTS A. Pre-Embedding Labeling B. Post-Embedding Procedures C. Cryoultramicrotomy Technique D. Negative Staining Procedures III. Immunoglobulins A. Protein A and Protein G Techniques B. Polyclonal and Monoclonal Antibodies IV. Common Immunolabeling Techniques for Electron Microscopy A. Immunoferritin B. Immunoperoxidase Techniques C. Immunogold Techniques References Chapter 15 Autoradiography 321 I. History II. Purpose III. Theoretical Aspects A. Detection of Radioactivity B. Types of Particles C. Nuclear Emulsions D. Determination of Isotope Dose Level E. Rules for Autoradiography F. Light Microscopy Autoradiography References Chapter 16 Computer-Assisted Imaging I. Purpose II. Resolution and Discrimination III. Image Processing IV. Morphometric Analysis V. Stereology VI. Computer-Assisted Analysis of Movement References Chapter 17 Scanning Tunneling Microscopy and Its Derivatives 341

15 CONTENTS Appendix A Laboratory Safety xvii 345 Appendix B Literature Sources for Electron Microscopy I. Atlases II. Journals III. Society Publications IV. Book Series V. NIH Resources for Intermediate Voltage Electron Microscopy (IVEM) Appendix C Electron Microscopy Equipment and Supplies 349 I. Expendable Supplies and Small Equipment II. Electron Microscopes III. Diamond Knives IV. High-Vacuum Pumps V. Ultramicrotomes VI. Equipment for Cryotechniques VII. Sputter Coaters and Vacuum Evaporators Index

16 Biological Electron Microscopy Theory, Techniques, and Troubleshooting