Biological Electron Microscopy Theory, Techniques, and Troubleshooting 2nd Edition Biological Elecfron Microscopy Theory, Techniques, and Troubleshooting 2nd Edition J. Michael 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 2003047709 ISBN 978-1-4613-4856-6 © 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. 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. 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 parti cular 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 instru ments 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 structure function 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 con cerning 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 instrumen tation 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 pro cedures 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 pro vided 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 intimidat ing 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 with out 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), high resolution scanning electron microscopes, and environmental scanning electron microscopes (ESEMs) have become easier to use, more reliable, and are more commonly found on university cam puses 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 hard ware 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 spe cific methods section at the end of selected chapters, however, is a major change from the first edi tion 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 par ticular, 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 confo cal 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 obser vations 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 asso ciated 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 study ing cells and tissues. We are devoted to the concept that understanding structure/function rela tionships 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 min imum, 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 spe cific 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 sam ple preparation procedures will serve to introduce you to a set of disciplines associated with cyto logical 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 cyto logical 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 1 I. Physical Fixation Techniques 1 II. Traditional Chemical Fixation 2 III. Buffers 24 IV. Dehydration 29 V. Embedding Media 31 VI. Examination of Tissues Prepared with a Variety of Fixatives and Buffers 36 VII. A Quasi-Universal Fixation, Dehydration, and Embedment Schedule Successfully Used for Organisms from the Five Major Kingdoms of Life 40 References 72 Chapter 1 Techniques 74 Making Dilutions 74 Diluting Stock Solutions 74 Preparing Stock Solutions to Be Mixed Together to Achieve Specific Final Concentrations in a Working Fixative Solution 74 A Routine Fixation and Embedding Schedule for Transmission Electron Microscopy Samples (Tissues or Cells) 74 Preparation of Primary (Aldehyde) Fixatives 80 Preparation of Osmium (Osmium Tetroxide, Osmium Tetraoxide) 81 Buffering Systems 85 Cacodylate Buffers 86 Phosphate Buffers 87 Tris-HCl Buffer 89 Sodium Acetate Buffer 90 Dulbecco's Phosphate-Buffered Saline 91 Resin Formulations 92 Spurr Resin 93 PolylBed 812 Resin 96 SPI-Pon 812 97 Araldite 6005 Resin 99 Mollenhauer's EponiAraldite Resin (Adapted for Use with Epon Substitutes) 100 London Resin Co. (LR) White Resin 101 Lowicryl and LR Gold Resins 103 PEG Method for TEM Sections 104 JB-4™ (Glycol Methacrylate) Techniques for High-Resolution Light Microscopy 105 Agar Embedment of Cell Suspensions or Subcellular Particulates for TEM 107 Preparing Adherent Tissue Culture Monolayers in Situ for TEM 109 xi XII Contents Flat Embedding of Cell Cultures Grown on Permanox® Tissue Culture Dishes for TEM III Preparation of Buffy Coats for TEM 111 Sperm Fixation 114 Central Nervous System Fixation (Brain, Spinal Cord) 116 Using Vacuum to Help Wet Fungal, Plant, or Insect Samples during Primary Fixation 117 Simultaneous Glutaraldehyde/Osmium Fixation for Protozoan Samples or Samples with a Large Amount of Lipid 118 Killing Cells Prior to Chemical Fixation 119 Flat Embedding on Microscope Slides 120 Procedure for Deparaffining Samples 122 Chapter 2 Cryotechniques 125 I. History 126 II. Purpose 127 III. Cryogens 128 IV. Safety Precautions 129 V. Freezing Methods 129 VI. Uses of Frozen Specimens 135 VII. Artifacts and Their Correction 146 References 149 Chapter 2 Techniques 150 Cryoultramicrotomy for Structural Examinations or Consequent Immunolabeling 150 Chapter 3 Ultramicrotomy 153 I. Ultramicrotomes 153 II. Knives 155 III. Block Trimming 157 IV. Ultrarnicrotomy Working Area 158 References 158 Chapter 3 Techniques 159 Making a Section Retrieval Loop 159 Making a Section Manipulation Tool 160 Making a Locking Ring for Forceps 161 Making Glass Knives 161 Making Glass Knife Boats 164 Glass Knife Storage 164 Block Trimming 165 Sectioning Procedures 167 Semi thin Sections 170 Grid Selection 171