Handbook of Cryo-Preparation Methods for Electron Microscopy (Methods in Visualization)

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Author: Annie Cavalier | Daniele Spehner | Bruno M. Humbel

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HANDBOOK OF

Cryo-Preparation Methods for Electron Microscopy

© 2009 by Taylor & Francis Group, LLC

Methods in Visualization Series Editor: Gérard Morel

Genome Visualization by Classic Methods in Light Microscopy Jean-Marie Exbrayat Handbook of Cryo-Preparation Methods for Electron Microscopy Annie Cavalier, Danièle Spehner, and Bruno M. Humbel Imaging of Nucleic Acids and Quantitation in Photonic Microscopy Xavier Ronot and Yves Usson In Situ Hybridization in Electron Microscopy Gérard Morel, Annie Cavalier, and Lynda Williams In Situ Hybridization in Light Microscopy Gérard Morel and Annie Cavalier PCR/RT -PCR In Situ Light and Electron Microscopy Gérard Morel and Mireille Raccurt Visualization of Receptors: In Situ Applications of Radioligand Binding Emmanuel Moyse and Slavica M. Krantic


Methods in Visualization Series

HANDBOOK OF

Cryo-Preparation Methods for Electron Microscopy

Edited by

Annie Cavalier Danièle Spehner Bruno M. Humbel


Painting on the cover by Annie Cavalier

CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2009 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-13: 978-0-8493-7227-8 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http:// www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Handbook of cryo-preparation methods for electron microscopy / editor, Annie Cavalier, Daniele Spehner, and Bruno M. Humbel. p. ; cm. -- (Methods in visualization) Includes bibliographical references and index. ISBN 978-0-8493-7227-8 (hardcover : alk. paper) 1. Cryobiology--Methodology. 2. Electron microscopy--Methodology. I. Cavalier, Annie. II. Spehner, Daniele. III. Humbel, Bruno M. IV. Title. V. Series. [DNLM: 1. Cryopreservation--methods. 2. Microscopy, Electron--methods. QY 95 H236 2008] QH324.9.C7H36 2008 570.28’25--dc22 Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com


2008016244

V

SERIES PREFACE The second half of the 20th century was a period of intense exploration into the anatomy of the cell. After the ground-breaking discovery of a whole new world of structural complexity, the period between about 1950 and 1970 was characterized by the further development of the electron microscope, and specimen preparation methods were redefined so that cells could be studied at the organelle level, then that of single molecular units. During the 1970s, correlating structure and function was the most productive area of cell biology. Electron microscopy was applied to cytochemical reactions developed for use with light microscopy, but also to histochemical reactions, immunolabeling and autoradiography. The pioneers of the 1960s were already studying individual molecules within cells, and by the 1970s these methods had gained wide acceptance. Since the 1960s, the most effective method for exploring submicroscopic structures has been ultramicrotomy. But the preparation steps carried out in conventional ultramicrotomy, i.e., chemical fixation using osmium tetroxide, chemical solvents and resin embedding, are harmful to structural conformation. In order to preserve the integrity of molecules in sections, researchers such as Leduc,1,2 Pease3,4 and their co-workers introduced water-miscible macromolecules as embedding material. This technique proved effective in enzyme digestion5,6 and the immunochemical labeling of a small number of antigens,7 and Bernhard’s group adapted it for use with sections of frozen aldehyde-fixed tissue.8-10 Finally, Tokuyasu introduced inert cryoprotectants, such as sucrose, into specimens in infusions before freezing so as to improve their plasticity, and the dry, frozen sections were collected on droplets of saturated sucrose.11,12 For x-ray analytical studies, no suitable flotation fluid has yet been found onto which frozen sections can be cut. Dry sectioning systems13,14 are therefore used, without chemical fixation. Electron microscopy, while presenting the advantages associated with frozen sections of unfixed and unembedded material, provides little ultrastructural detail, due to the damage caused by the freezing process. “A very serious problem in any morphological study of frozen tissue is the ice crystal damage that occurs during the initial freezing and subsequent recrystallization.”13 Christensen14 found that rapid freezing minimized the size of the ice crystals. A number of other ways of minimizing these artifacts have been proposed. Chemical fixation or cryoprotection, for example, can reduce them. But only very rapid freezing, using specifically designed equipment, will result in satisfactory frozen preparations. In 1981, Allakhverdov and Kuzminykh15 wrote: “Well-known advantages of specimen preparation by cryomethods are accompanied by some disadvantages, resulting mainly from the inadequate level of presently existing laboratories and instruments.” These days, there are laboratories that specialize in cryomethods, and state-of-the-art equipment is now available for the preparation and observation of frozen specimens. Thin-film vitrification is a practical freezing technique that does not need very specialized equipment, but its application is limited by the fact that the material to be observed has to be thin enough for direct observation by transmission electron microscopy. Cryoultramicrotomes and electron microscopes are now capable of dealing with larger and/or thicker frozen samples, resulting in ever better observations and a growing range of applications.


VI For the moment, cryomicroscopy using water vitrification is the method of choice for research in the field of structural biology, combined with x-ray analysis and particle visualization (e.g., nanoparticles, macroparticles, viruses, filamentary structures). Vitrification in cells and tissue can attain a depth of 10 µm, and it is now possible to observe intact cells and tissue in their natural state. In the words of Dubochet16 — though indeed Fernandez-Moran, in the early 1950s,17 might have said much the same thing — “That is what cryo-EM of vitreous sections (CEMOVIS) does.” Gérard Morel Series Editor 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Leduc, E.H. et al. The use of water-soluble glycol-methacrylate in ultrastructural cytochemistry, J. Roy. Microscop. Soc., 81, 119, 1963. Leduc, E.H. and Holt, S.J. Hydroxy-propyl methacrylate, a new water-miscible embedding medium for electron microscopy, J. Cell Biol., 26, 137, 1965. Pease, D.L. Eutectic ethylene glycol and pure propylene glycol as substitution media for dehydratation of frozen tissue, J. Ultrastructural. Res., 21, 75, 1967. Pease, D.L. and Peterson, R.G. Polymerisable glutharaldehyde-urea mixture as polar, water-containing embedding media, J. Ultrastructural. Res., 41, 133, 1972. Bernhard, W. and Granboulan, N. The fine structure of the cancer cell nucleus, Exp. Cell Res. Suppl, 9, (19), 19, 1963. Granboulan, N. and Granboulan, P. Ultrastructure cytochemistry of the nucleolus. Demonstration of the chromatin inside the nucleolus, Exp. Cell Res., 34, 71, 1964. Kawarai, Y. and Nakane, P.K. Localization of tissue antigens on the ultrathin sections with peroxidase-labeled antibody method, J. Histochem. Cytochem., 18, 161, 1970. Bernhard, W. and Leduc, E.H. Ultrathin frozen sections. I. Methods and ultrastructural preservation, J. Cell Biol., 34, 757, 1967. Bernhard, W. and Viron, A. Improved techniques for the preparation of ultrathin frozen sections, J. Cell Biol., 49, 731, 1971. Leduc, E.H. et al. Ultrathin frozen sections. II. Demonstration of enzymatic activity, J. Cell Biol., 34, 773, 1967. Painter, R.G. et al. Immunoferritin localization of intracellular antigens: The use of ultracryotomy to obtain ultrathin sections suitable for direct immunoferritin staining, Proc. Nat. Acad. Sci., USA, 70, 1649, 1973. Tokuyasu, K.T. A technique for ultracryotomy of cell suspensions and tissues, J. Cell Biol., 57, 551, 1973. Appleton, T.C. A cryostat approach to ultrathin "dry" frozen sections for electron microscopy: A morphological and x-ray analytical study, J. Microsc., 100, 49, 1974. Christensen, A.K. Frozen thin sections of fresh tissue for electron microscopy, with a description of pancreas and liver, J. Cell Biol., 51, 772, 1971. Allakhverdov, B.L.and Kuzminykh, S.B. Some aspects of developing instruments for cryomethods, Acta Histochem., 23, 75, 1981. Al-Amoudi, A. et al. Cryo-electron microscopy of vitreous sections, EMBO J., 23, 3583, 2004. Fernandez-Moran, H. Application of the freezing sectioning technique to the study of cell structure with the electron microscope, Ark. Fys., 4, 471, 1952.


VII

ACKNOWLEDGMENTS

This handbook was initiated by Annie Cavalier and Danièle Spehner as part of a Centre National de la Recherche Scientifique (CNRS) effort in France (GDR 2368) to promote cryo-electron microscopy. Bruno Humbel, in Holland, enthusiastically joined the team. We are indebted to the contributors who have made it come true. It was not such an easy task explaining tricks of the trade, routinely carried out by each of them, but not so readily put into words. Dr. Gerard Morel provided his guidance throughout and we are very grateful to him. We also thank Profs. Daniel Boujard and Gilles Salbert who have encouraged the project and ensured a propitious environment. Our thankfulness extends to Dr. Patrick Schultz and Prof. Arie Verkleij who believed in the usefulness of such a handbook and supported us during its elaboration. Bruno Humbel would also like to thank the European Network of Excellence (NoE), FP6: “Three-Dimensional Electron Microscopy” and the Dutch Cyttron Consortium, “A Window on the Molecular Machinery of Life,” for support. We express our gratitude to Dr. Robert Drillien who not only overviewed the use of the English language, but also shared his insight on the contents of the book. This project has also benefited from the generous help of Emmanuelle Guiot who kindly performed the layout of the entire handbook; her participation was most valuable. Sandrine Pawlicki kindly constructed the book’s temporary Web site for which we are thankful. Last but not least, we would like to thank Christian Cavalier who offered us his home and his hospitality for a few hectic but very enjoyable weeks during finalization of the manuscripts. Annie Cavalier Danièle Spehner Bruno M. Humbel


IX

THE AUTHORS Annie Cavalier, graduate of the University Claude Bernard-Lyon 1, is currently an engineer in Biology in a CNRS laboratory at the University of Rennes I, France. Annie Cavalier is a member of the French Society of Microscopy (SFµ). She was actively involved in the organization of the 13th International Congress of Electron Microscopy (Paris, July 1994), and the first Congress of the French Society of Microscopy (Rennes, June 1996). She is secretary of the GUMP (Groupement des utilisateurs des microscopes Philips-FEI; a unique association that brings together users of FEI-made electron microscopes). Annie Cavalier has regularly given lectures and practical courses for training in the field of cytology at the ultrastructural level. She has edited two technical books and contributed to numerous scientific papers. Until recently, her research interests have focused on the structural and functional characterization of water channels (aquaporins) and glycerol facilitators. Her current research concerns the assembly and dynamics of large macromolecular complexes by means of electron tomography. Danièle Spehner obtained her Ph.D in Biology from the University Louis Pasteur in Strasbourg, France, while working at the Virology Laboratory of the Medical School in Strasbourg. After a few years at Transgene, a biotechnology company, where she developed new vaccines based on poxvirus recombinants, she returned to fundamental research at the French Institute of Health and Medical Research (INSERM). She is currently an engineer at the Institute of Genetics and Molecular and Cellular Biology (IGBMC) in Illkirch, France. Early in her career, she became interested in electron microscopy to study poxviruses as such or recombinant poxviruses expressing foreign proteins. She acquired a passion for cryo-electron microscopy in biology after meeting Professor Hellmut Sitte at a workshop in Seefeld (Austria) and, later on, Bruno Humbel. She transmitted her passion and her know-how through cryo or immuno workshops to the Alsatian community and throughout France. Danièle’s current research focuses on the use of electron tomography and cryo-electron tomography of vitreous cryo-sections (Cemovis) to analyze poxvirus morphogenesis and the cell nucleus. Bruno M. Humbel graduated in Biochemistry at the Federal Institute of Technology (ETH), Zürich, Switzerland. He was privileged to do his Ph.D with Dr. Martin Müller in the group of Prof. Dr. Hans Moor at the Institute of Cell Biology, ETH. After a postdoc period of four years at the Max-Planck-Institute for Biochemistry in Munich, he joined the lab for Electron Microscopy and Structure Analysis at Utrecht University in the group of Prof. Dr. Arie J. Verkleij, where he currently holds the position of an associate professor. Bruno Humbel’s main interest is to develop preparation methods for (electron) microscopy, which not only allow a glimpse into life at high resolution, but also enable identification and localization of the machinery of life within cells. The aim is to visualize the living cell at low resolution and to zoom in to analyze its ultrastructure at high resolution: The gateway to in situ biological nanostructures. Recently, his research focuses on correlative microscopy to introduce the FIB/SEM technology into life sciences and the application of a newly developed integrated laser electron microscope (ILEM). He is teaching cryo-techniques and immunolabeling at Utrecht University and in different workshops in Europe and Asia.


X

CONTRIBUTORS AL-AMOUDI Ashraf EMBL HEIDELBERG Germany

CHRÉTIEN Denis UMR CNRS 6026, Equipe TIPs Campus de Beaulieu Université de Rennes 1 RENNES France

ARNAL Isabelle UMR CNRS 6026, Equipe TIPs Campus de Beaulieu Université de Rennes 1 RENNES France

CRUCIFIX Corinne Institut de Génétique et de Biologie Moléculaire et Cellulaire Inserm U596, CNRS UMR7104 Université Louis Pasteur de Strasbourg ILLKIRCH France

BAUMEISTER Wolfgang Max-Planck-Institut für Biochemie Abteilung Strukturbiologie MARTINSRIED bei München Germany

DE CARLO Sacha MCDB 347 University of Colorado, BOULDER Colorado USA

BORDAT Christian INRA-CRJ NURELICE, Bât. 230 Domaine de Vilvert JOUY EN JOSAS France

DE HAAS Felix FEI Company EINDHOVEN The Netherlands

BOUCHER-MARQUIS Cédric University of Colorado Dept. of Molecular Cellular and Developemental Biology, Porter Science, BOULDER Colorado USA

DUBOCHET Jacques Centre de Microscopie Laboratoire d’Analyses Ultrastructurales Université de Lausanne LAUSANNE Switzerland

BRON Patrick UMR CNRS 6026, Equipe SDM Campus de Beaulieu Université de Rennes 1 RENNES France

EDELMANN Ludwig Anatomie und Zellbiologie, Universität des Saarlandes, HOMBURG/Saar Germany

CAVALIER Annie UMR CNRS 6026, Equipe SDM Campus de Beaulieu Université de Rennes 1 RENNES France

ELTSOV Mikhail EMBL HEIDELBERG Germany


XI FREDERIK Peter M. Maastricht University EM Unit / Pathology MAASTRICHT The Netherlands

KAECH Andres Center for Microscopy and Image Analysis at the University of Zurich ZÜRICH Switzerland

FUKAZAWA Yugo Division of Cerebral Structure National Institute for Physiological Sciences, Myodaiji OKAZAKI Japan

LEIS Andrew Max-Planck-Institut für Biochemie Abteilung Strukturbiologie MARTINSRIED bei München Germany

GEERTS Willie J. C. Electron Microscopy & Structure Analysis Cellular Architecture  Dynamics Utrecht University UTRECHT The Netherlands

MALLETER Marine Laboratoire de pharmacologie marine Faculté des Sciences de Nantes NANTES France

GUERQUIN-KERN Jean Luc Institut Curie Recherche/INSERM U 759 Laboratoire Microscopie Ionique Bat.112 Centre Universitaire ORSAY France

MASUGI-TOKITA Miwako Division of Cerebral Structure National Institute for Physiological Sciences, Myodaiji OKAZAKI Japan

GRUSKA Manuela Max-Planck-Institut für Biochemie Abteilung Strukturbiologie MARTINSRIED bei München Germany

MÉSINI Philippe J. Institut Charles Sadron-CNRS-ULP STRASBOURG France

HAGIWARA Akari Division of Cerebral Structure National Institute for Physiological Sciences, Myodaiji OKAZAKI 444 Japan

MOREL Gérard UMR 5123 CNRSUniversité Claude Bernard-Lyon 1 VILLEURBANNE France

HUMBEL Bruno M. Electron Microscopy & Structure Analysis Cellular Architecture  Dynamics Utrecht University UTRECHT The Netherlands

PAPAI Gabor Institut de Génétique et de Biologie Moléculaire et Cellulaire Inserm U596, CNRS UMR7104 Université Louis Pasteur de Strasbourg ILLKIRCH France


XII SCHMUTZ Marc Institut Charles Sadron-CNRS-ULP STRASBOURG France

STUDER Daniel Institute für Anatomy University of Bern BERN Switzerland

SCHULTZ Patrick Institut de Génétique et de Biologie Moléculaire et Cellulaire Inserm U596, CNRS UMR7104 Université Louis Pasteur de Strasbourg ILLKIRCH France

TARUSAWA Etsuko Division of Cerebral Structure National Institute for Physiological Sciences, Myodaiji OKAZAKI Japan

SCHWARZ Heinz Max-Planck-Institut fuer Entwicklungsbiologie TÜBINGEN Germany

THOMAS Daniel UMR CNRS 6026, Equipe SDM Campus de Beaulieu Université de Rennes 1 RENNES France

SHIGEMOTO Ryuichi Division of Cerebral Structure National Institute for Physiological Sciences, Myodaiji OKAZAKI Japan

VAN DONSELAAR Elly Electron Microscopy & Structure Analysis Cellular Architecture  Dynamics Utrecht University UTRECHT The Netherlands

SITTE Hellmuth HOMBURG-SAAR Germany or SEEFELD in Tyrol Austria

VANHECKE Dimitri Institute für Anatomy University of Bern BERN Switzerland

SPEHNER Danièle Institut de Génétique et de Biologie Moléculaire et Cellulaire Inserm U596, CNRS UMR7104 Université Louis Pasteur de Strasbourg ILLKIRCH France

VERKLEIJ Arie J. Electron Microscopy & Structure Analysis Cellular Architecture  Dynamics Utrecht University UTRECHT The Netherlands

STIERHOF YorkDieter Zentrum für Molekularbiologie der Pflanzen (ZMBP). Elektronenmikroskopie, Universität Tübingen TÜBINGEN Germany

VONCK Janet Max-Planck-Institute of Biophysics Department of Structural Biology FRANKFURT am Main Germany

STORMS Marc M.H. FEI Company EINDHOVEN The Netherlands

ZUBER Benoît MRC Laboratory of Molecular Biology CAMBRIDGE United Kingdom


XIII

TABLE OF CONTENTS Introduction

XVII

Abbreviations

XIX

Chapter 1 Vitreous Water Jacques Dubochet

1

Part I  Cryo-Fixation Methods Chapter 2 Slam-Freezing, Metal-Mirror Freezing Danièle Spehner and Ludwig Edelmann  Chapter 3 Plunge-Freezing (Holey Carbon Method) Sacha De Carlo

17

Chapter 4 Controlled Vitrification Peter M. Frederik, Felix de Haas and Marc M.H. Storms

69

Chapter 5 BAL-TEC HPM 010 High-Pressure Freezing Machine Andres Kaech

101

Chapter 6 High-Pressure Freezing LEICA EMPACT Dimitri Vanhecke and Daniel Studer

129


49

XIV

Part II  Cryo-Electron Microscopy Chapter 7 Frozen-Hydrated Macromolecules for Structural Analysis Corinne Crucifix, Gabor Papai and Patrick Schultz

159

Chapter 8 Two-Dimensional Crystals Patrick Bron and Janet Vonck

191

Chapter 9 Cryo-Negative Staining Sacha De Carlo

219

Chapter 10 Vitrification of Dynamic Microtubules Isabelle Arnal, Marine Malleter and Denis Chrétien

237

Chapter 11 CEMOVIS: Cryo-Electron Microscopy of Vitreous Sections Jacques Dubochet, Ashraf Al-Amoudi, Cédric Bouchet-Marquis, Mikhail Eltsov and Benoît Zuber

259

Chapter 12 Cryo-Electron Tomography Andrew Leis, Manuela Gruska and Wolfgang Baumeister

291

Part III  Low-Temperature Embedding Chapter 13 Freeze-Substitution Bruno M. Humbel

319

Chapter 14 Cryo-Fixation, Freeze-Substitution, Rehydration and Tokuyasu CryoSectioning YorkDieter Stierhof, Elly van Donselaar, Heinz Schwarz, Bruno M. Humbel

343

Chapter 15 Freeze-Drying and Embedding of Biological Material Ludwig Edelmann

367


XV

Part IV  Freeze-Fracture and Metal Shadowing Chapter 16 The Shadow of Hydrated Biological Specimens Daniel Thomas

391

Chapter 17 Cryo-Fracture of Self-Assembled Systems in Organic Solvent Marc Schmutz and Philippe J. Mésini

411

Part V  Analysis Chapter 18 Progressive Lowering of Temperature for Immunolabeling and in situ Hybridization Annie Cavalier and Danièle Spehner

433

Chapter 19 Cryo-Sectioning According to Tokuyasu Bruno M. Humbel and YorkDieter Stierhof

467

Chapter 20 Cryo-Preparation Procedures for Elemental Imaging by SIMS and EFTEM Christian Bordat and Jean-Luc Guerquin-Kern

499

Chapter 21 Correlative Light and Electron Microscopy Heinz Schwarz and Bruno M. Humbel

537

Chapter 22 SDS-digested Freeze-Fracture Replica Labeling (SDS-FRL) Yugo Fukazawa, Miwako Masugi-Tokita, Etsuko Tarusawa, Akari Hagiwara and Ryuichi Shigemoto

567

Chapter 23 Immunolabeling of Ultrathin Sections with Enlarged 1 nm Gold or QDots YorkDieter Stierhof

587

Chapter 24 3-D Electron Tomography of Cells and Organelles Willie J. C. Geerts, Bruno M. Humbel and Arie J. Verkleij

617

Final Considerations Hellmuth Sitte

651

Glossary

657


XVII

INTRODUCTION The purpose of this handbook is to provide guidance to newcomers to the field who wish to learn and possibly apply the methods it describes. Of course, no biologist knows every technique in his specialty and this also pertains to those using electron microscopy, so we hope that our fellow colleagues will also find this book useful for one application or another. The chapters have been written by experts in the field, in some instances the very inventors of the method, who have taken care to explain the history behind the techniques they describe and how they are most readily carried out today. Whenever possible, clear step-by-step recipes are presented and the tools and ingredients of the methods, as well as where they can be purchased, are listed. After a quick glance at the contents one will realize that this handbook goes well beyond the preparation methods the title suggests and into the realm of cryo-electron microscopy analysis itself, hence the numerous illustrations from the contributors’ own laboratories. Most biologists have their own idea of what cryo-electron microscopy is all about and this notion may be strongly influenced by one’s own experience as well as the fashion of the times. It has long been realized that freezing specimens is a nearly perfect way to maintain biological samples in a state as close as possible to their native state. Over the years many different methods have been devised to freeze samples and each of them has been presented with advice on how they should be applied and to what kind of material. The ultimate achievement in this field is certainly the ability to embed samples into a somewhat mysterious form of ice known as vitreous water or crystal-free ice. Today, there are numerous ways to take advantage of this technological feat. Whereas some methods aim at perfect preservation for fine structural analysis, others use vitreous water as an intermediate step for in situ localization of biological molecules and ions. Sophisticated methods of resin embedding subsequent to removal of vitreous water, as well as freeze-fracture, have become valuable tools for numerous applications as illustrated in this handbook. Ice also turned out to be a convenient medium for embedding and sectioning, as has been so exquisitely exemplified by the so-called Tokuyasu method, and dealt with in several chapters herein. This and the so-called hybrid methods, freezesubstitution and freeze-drying, have been found to be ideal intermediates to achieve highly efficient immunolabeling, so critical for determining the molecular geography inside tissues and cells. On the other hand, such methods have gained considerably from parallel developments including new ways to stain samples and label them. This opus would have been deficient if it had not also dealt with one of today’s most promising developments in cryo-electron microscopy, namely cryo-electron tomography. Although such tools would deserve an entire book in their own right, the chapters herein should provide a useful overview of the topic. The pace of cryo-electron microscopy has been accelerating in recent years and the methods available are constantly evolving. It is our wish that this handbook be a useful aid to the scientific community for current and future endeavors, and especially for novices to show the beauty of cryo and encourage them to take up the challenge.


XIX

ABBREVIATIONS 2-D 3-D ART CCD CEMOVIS CET CFD CIA CMC Cryo-ET Cryo-EM CS CTF EELS EF EFTEM ESI ET FD FEG FF FRL FS HAADF HC-PRO HPF LR LTE MCS MES MMF MS medium PLT Pt/C QD RTS SIMS SIRT WBP θ


 Two-Dimension/two-Dimensional  Three-Dimension/three-Dimensional  Algebraic Reconstruction Technique  Charge Coupled Device (camera)  Cryo-Electron Microscopy Of Vitreous Sections  Computerized electron tomography  Cryo-sorption Freeze-Drying system  Cutting-Induced Amorphous water  Critical Micelle Concentration  Cryo-Electron Tomography  Cryo-Electron Microscopy (of vitrified specimens)  Cryo-sectioning according to Tokuyasu  Contrast Transfer Function  Electron Energy-Loss Spectrum  Energy Filtering  Energy Filtered Transmission Electron Microscope  Electron Spectroscopic Imaging  Electron Tomography  Freeze-Drying  Field Emission Gun  Freeze-Fracturing/Freeze-Fracture  Freeze-fracture Replica Labeling  Freeze-Substitution  High-Angle Annular Dark-Field  Helper Component PROteinase  High-Pressure Freezer, High-Pressure Freezing  London Resin  Low-Temperature Embedding  Membrane Contact Side  Morpholino Ethane Sulfonic acid  Metal Mirror Freezing  Murashige and Skoog medium  Progressing Lowering of Temperature  Platinum used in conjunction with carbon  Quantum dot  Rapid transfer system  Secondary-Ion Mass Spectrometry  Simultaneous Iterative Reconstruction Technique  Weighted Back-Projection  Angle of shadowing


3

Vitreous Water

CONTENTS

1.

INTRODUCTION ................................................................................................. 5

2.

WATER IS REMARKABLE................................................................................ 5 2.1. 2.2.

Ice floats ....................................................................................................... 5 Water has a high specific heat ...................................................................... 6

3.

VITREOUS WATER IS STRANGE.................................................................... 7

4.

VITREOUS WATER MADE SIMPLE ............................................................... 9 4.1. 4.2. 4.3.

Vitrification is a good preservation method.................................................. 9 What is vitrification and devitrification? ...................................................... 9 How can vitrification be achieved? ............................................................ 10

5.

OBSERVED RESULTS ...................................................................................... 12

6.

REFERENCES .................................................................................................... 14


Vitreous Water

5

1. INTRODUCTION NASA would like to extend biological studies to other worlds and consequently makes every effort to look for water on other planets. For us, on earth, aiming at a better understanding of our earthly life, water is also of primary concern. It is the major constituent of all living organisms; it is the medium in which life takes place and it directly participates in most actions that make life work. Nevertheless, water is frequently neglected and it remains poorly understood in several essential aspects. The systematic exclusion of water as an object of investigation during the first 50 years of electron microscopy certainly contributed to the bias against it. Times have changed, however; for two decades now water has regained the central role it always had in nature. This book tells the story of its changed fortunes.  Many electron microscopists know little about water and even less about the strange properties of vitreous water. The present chapter aims at familiarizing them with the substance with which they are working. The literature on vitreous water is dangerously abundant and it is rather difficult for those who want to gain an understanding of it from first principles.1-3 Most of our review of 1988 still holds.4 For pleasure, we recommend the Bibliography of Water by Philip Ball.5 A recent review by Angell6 sums up the present situation, gives abundant references to the literature and, for the first time, incorporates the observations of cryo-electron microscopy with the bulk of data from the water field. In the following presentation, we aim to examine some fundamental questions about water and vitreous water. This simple presentation — hopefully not simplistic — will probably raise some specialists’ eyebrows. My excuse is my own incompetence. As a long-time tourist of “Waterland” I hope that it may offer a useful understanding for practitioners.

2. WATER IS REMARKABLE Chapters on water traditionally start with a list of the unusual properties that make water remarkable. We will consider only two of them: ice floats and liquid water has a high specific heat. These two properties are easy to grasp and they are convenient proxies for all the others.

2.1. Ice floats When a room is full, a good way to gain space is to make order. The contrary holds for water for which the disordered liquid form is 8 percent more dense than hexagonal ice, the ordered form we are used to. How can this be? Very simply, it all depends on how order is made. For example, in your home, you will use up a lot of space if you place only one book per shelf. This is similar to what happens with water.


6

Handbook of Cryo-Preparation Methods for Electron Microscopy

In a plane, one circle can touch up to six identical nonoverlapping neighbours (see Figure 1.1A). In space, identical spheres can accommodate up to twelve nearest neighbours. In hexagonal ice or cubic ice, each water molecule has only four next neighbours. Why so few? Because of hydrogen bonds! Each water molecule offers two and accepts two in a tetragonal symmetry (see Figure 1.1B). The rule of four can be extended to the neighbours and to the neighbours of the neighbours without limit. The result is ice. Two forms are possible: cubic ice (a form with the same density frequently encountered in cryo-electron microscopy) when the bonds of neighbouring tetrahedron are coplanar, or hexagonal ice in which tetrahedrons are rotated by 60° with respect to each other, as is the case in Figure 1.1C. With only four instead of twefve next neighbours, ice is full of holes. Hydrogen bonds can be seen as long sticks holding the molecules at a distance rather than glue holding them tightly together. Because of the large amount of free space between water molecules, other arrangements involving bending and stretching of hydrogen bonds are possible. The result is several other forms of crystalline ice. In most cases, they are only stable under pressure. The hydrogen bond is not very strong. It tends to break when temperature increases. Above 100°C at 1 atm, it cannot even hold the water molecules together, and water vapour becomes the most stable form. At intermediate temperatures, hydrogen bonds constantly break and reform, thus enabling any molecule to fall down for an instant, into the hole, closer to its neighbour. The structure is disordered, the density is higher; this is liquid water.

2.2. Water has a high specific heat Everything that can shake or move does so at the molecular level. This is temperature. Each of these things that can shake or move is called a degree of freedom. It is remarkable that each degree of freedom contains on average the same amount of energy  proportional to the temperature T:  = ½ kT, where the proportionality factor k is the Boltzmann’s constant, 1.38.10-23 J/oC. Everything else remaining equal, warming means increasing the amount of energy contained in each degree of freedom. The specific heat — the amount of heat required to warm one gram by one degree Celsius — is therefore directly proportional to the number of degrees of freedom n. n is a remarkable number. It tells us how complicated a system is. It tells us how many parameters must be introduced in any realistic model of the system. It tells us the magnitude of the task for those who have the ambition to understand it. We shall try an exercise. n is measured to be nine for water vapour. For each molecule, three degrees of freedom are required to specify its location in space (X, Y, Z) and three others for its orientation (3 Euler angles, three are left for internal vibrations of the molecule. This makes sense! What about ice, which has n = 8? As compared to water vapour, we expect the internal vibrations to be the same — that accounts for three degrees of freedom — but the six involved in location and orientation are lost because they are now nearly fixed in a crystal. So, where are the other five? Solid-state physics gives us the answer. These five are the collective vibrations of the crystal lattice; the molecules are allowed to move, but only in restricted cooperative movements. We come now to liquid water. What


Vitreous Water

7

do we expect? Internal vibrations remain; those are still three. Location and orientation: Molecules are quite free in liquid water, but not so much as in gas; it will be some number smaller than six, say, four. We are left with possible cooperative vibrations as in the crystal, but water is not known for its crystalline sound; it goes “splash” not “cliiing”; so instead of five, we can perhaps save three. And thus we have: n = 3 + 4 + 3 =10. But no, this cannot be! The specific heat of liquid water is more than twice that of vapour or of ice; n = 18. That is eight degrees of freedom more than our guess. We can add one or two more degrees for positional freedom and better collective vibrations, but how to reach eight? Where are the missing degrees of freedom? Apparently, nobody knows for sure. They will probably have to be found in some more cooperative interactions resulting in a much richer conformational space. Certainly, the years to come will bring a solution to the problem of the missing degrees of freedom. I like to guess that the solution will have interesting consequences in various fields where water is important — probably in biology also. Certainly, it is wise to keep an eye on water. 

3. VITREOUS WATER IS STRANGE Vitrification of water is an old idea, loaded with hopes and dreams. Imagine cooling water — or an organism — in such a way that it becomes immediately immobile. It is frozen time, suspended life. A science was born out of these ideas and the state of suspended animation got its name: the vitreous state.7 It turned out, however, that vitrification was terribly difficult to achieve except in the case of concentrated solutions. Even worse, the argument was made that vitrification of pure water is fundamentally impossible. The discovery, at the beginning of the 1980s, that the vitreous state can be obtained from liquid water8,9 was first greeted with scepticism, but because the procedure is simple and reproducible, some way must be found to reconcile theory and practice. We will explain why it is still a difficult task. The phase diagram of water sets the scene (see Figure 1.2). There is one domain for the liquid phase at the upper right, one domain for the vapour at the bottom, and one for ice on the left. We note that the vapour and liquid domains are not completely separated. The line goes to the so-called critical point and then stops. This is because there is not always a difference between these two phases. Above the pressure and temperature of the critical point, gas and liquid are all the same. On the contrary, ice is always separated from the domains of liquid and vapour. There is a good reason for the strict separation of the solid domain: The existence of a critical point between two phases means that it is possible to go continuously from one to the other. This cannot exist between the liquid, which has no symmetry, and hexagonal ice, which has hexagonal symmetry. An intermediate state with “just a bit of symmetry” makes no sense. At normal pressure, the most stable state is ice below 0°C, it is liquid between 0°C and 100°C and vapour is the normal state above 100°C. These are the conditions at which the free energy G is minimal as illustrated in Figure 1.3 for the liquid (A) and for the vapour region (B). This does not mean, however, that the liquid does not exist above 100°C; one can still warm it above this temperature provided it does not cross the potential barrier separating it from the vapour phase (see Figure 1.3 C). It is said to be in a metastable state.


8


Upon further warming, the advantage of the vapour phase over the liquid phase becomes greater, and the potential barrier separating them becomes smaller. The higher the temperature, the more difficult it is to keep the superheated liquid from escaping as vapour. Upon further warming, there is a point where the potential barrier becomes zero. Above this temperature, the liquid state just does not exist anymore. The line depicting this limit on the phase diagram is called the spinodal line (see Figure 1.2). An analogy may help to clarify the concept. My pen on the desk is in a metastable state; the stable state is when it lies on the floor. If the table is smaller, the pen can still be on the table though it will fall more frequently. If the table is removed, the question whether the pen is on the table or not ceases to make sense. Similarly, there is no question anymore about the liquid state after the spinodal line. However, the region just before the spinodal line is particularly interesting. Of course, it is difficult to explore it because the liquid takes every opportunity to vaporize. In this region, all the properties of the liquid become strange. A graph presenting the variation of any property of the superheated liquid always shows that something special is happening when the temperature approaches the critical line. The signature of the approaching catastrophe is characteristic. What is the freezing temperature of water? This trap question calls for the wrong answer. 0°C is not the freezing temperature of water at normal pressure; it is the melting temperature of ice! The freezing temperature of liquid water is somewhere below 0°C, whenever the liquid finds its way toward the more stable state of ice. This is an important difference because, in nature, it frequently happens that the temperature fluctuates around 0°C. It makes a lot of difference as to whether water freezes or not. The matter has been studied in detail and one basic fact stands out: Water resists undercooling at low temperatures pointing toward a spinodal line at around 45°C. The signature is the same as for superheated water. It explains why attempts to vitrify pure water were unsuccessful for so long, just like keeping the pen on the table does not work when there is no table. But this does not explain the finding in 1980 that ultrarapid cooling can vitrify liquid water! Perhaps, after all, there is no spinodal at 45°C and the strange behaviour of undercooled water must be explained differently? Soon after, another finding was made, complicating the situation even further. Under very high pressure (10,000 bar), and cooled at liquid nitrogen temperature, hexagonal ice crumbled into a High-Density Amorphous state (1.15 g/cm3 after restoration of normal pressure) poetically called HDA, which is clearly different from LDA, the Low Density Amorphous state (0.94 g/cm3) obtained by rapid cooling of liquid water.10 This could be explained by considering that disorder can take many forms from low to high density amorphous, just as the disorder in a room can correspond to any level of heaping. But as usual, water surprises us. When HDA is rewarmed slowly, it suddenly changes into something resembling LDA. The transformation cannot be ignored because the abrupt change of density results in large internal tensions, which make the block fly apart in small fragments. It was also observed by electron microscopy.11 Thus, it appears that amorphous water is disordered in at least two mutually exclusive states. In HDA, the disorder is organised in some way and it becomes unstable above 160°C, at which temperature the disorder must be organized in a different way. The big trouble is that nobody knows what makes the difference. Keep an eye on vitreous water, it could tell us important things about water!


Vitreous Water

9

4. VITREOUS WATER MADE SIMPLE What are the consequences of the above consideration for us, cryo-electron microscopists? For one thing, and as always in science, it tells us that we should remain vigilant because we do not know what we are observing. It could be that our vitrified specimens are fundamentally different from the original material. In them, water is perhaps organised differently. The future will tell.

4.1. Vitrification is a good preservation method The other thing we know is that vitrification is an excellent method for preserving biological material. It makes it possible to observe samples without going through the major transformations of chemical fixation, dehydration and staining. Even more important, vitrification is, by itself, a remarkably conservative procedure. This is demonstrated by a large body of evidence extending from cryo-preservation of embryos for in vitro fertilization, to cryo-x-ray diffraction for best preservation of atomic structures in delicate crystals. The quality and the coherence of the results obtained by cryo-electron microscopy over the last 20 years also contribute to the credibility of the method. 

4.2. What is vitrification and devitrification? In everyday life, and until we know more about vitreous water, cryo-electron microscopists can resist becoming depressed by following Occam’s recipe, adopting the simplest possible view of vitrification and of the vitreous state. In this view, vitrification is the process by which the viscosity of the sample is increased to such a high value that molecular movements become negligible before ice crystal formation has time to start. Any subsequent change is blocked unless considerable forces are applied. Typically, such forces may arise during cutting thin sections and during observation in the electron microscope. For a normal experimental time scale, the vitreous state holds as long as the temperature is well below 135°C. At this temperature, the movement of the water molecules becomes significant enough so that crystallisation takes place within minutes. This is the process of devitrification. It can take place at a slightly lower temperature if one waits long enough or if it is accelerated by electron irradiation. We note that ice formation by crystallisation from the cooling liquid is very different from ice formation by devitrification. In the first case, it takes place in the liquid medium where everything is mobile. As a consequence, the growing ice crystal, which only incorporates water molecules, rejects all the other constituents of the solution, such as salts, other solutes and macromolecules. Segregation takes place between pure water in the ice crystals and all the dehydrated remains of the biological material. Ice formation by devitrification, on the other hand, takes place under conditions of severely restricted mobility — just enough to allow water molecules to rearrange into ice crystals. Under these conditions, no large ramified hexagonal ice crystal is formed, but instead many compact, sub-µm cubic crystals appear.


10


They are easily recognized from the characteristic circles of the powder diffraction spectra. In spite of the fact that little is known about the devitrification process, it probably takes place with minimum rearrangement of the biological material. One can infer that the fine structure of a frozen biological specimen in which ice is formed by devitrification is better preserved than when ice grows from the liquid phase.12 These considerations are important for freeze-substitution (see Chapter 13), which is always performed well above the devitrification temperature. Substitution, therefore, deals with water in the form of crystalline ice. It probably makes a lot of difference whether this ice was formed by devitrification of a vitrified specimen or whether it was crystallized during cooling from the liquid phase. The best route is certainly to start with a vitrified sample.

4.3. How can vitrification be achieved? In principle, the method is simple. It suffices to cool the specimen so rapidly that water molecules are practically immobilized before an ice crystal starts to form. By chance, nucleation, the beginning of ice crystal formation, is not an easy task. Even billions of molecules take a while until, by chance, a group of them happens to be in the right conformation. Once a crystal has started, the road is clear; it produces its own heat, further amplifying its growth until all the available water is sequestrated. The question we may address is, therefore: What is the cooling speed required for vitrification?  The answer is: Nobody knows! Fortunately we know quite well what are the important parameters, and experience tells us how they can be combined to reach vitrification. The easiest way is to increase cooling speed by reducing the size of the specimen. Physics has found that cooling speed increases with the inverse square of the size of an object immersed in a good cryogen. Calculation13 tells us that, one cm deep in a piece of meat suddenly placed at liquid nitrogen temperature, the temperature drop is expected to be in the oC/sec range; at 0.1 mm from the surface, it will reach ca. 104 oC /sec and for a 200 nm thin layer, it is deduced to reach the staggering value of 1011 oC /sec. It is a big advantage for electron microscopists, and the green light towards cryo-electron microscopy, that vitrification is relatively easy when the specimens are of a size that is generally observed in a transmission electron microscope (TEM). Chapters 5 and 6 describe how it can be done. In general, it is considered that vitrification of pure water is not possible for dimensions in excess of some µm. Some µm are more than enough if the specimen is to be directly observed in a transmission electron microscope. Macromolecules, viruses, many isolated organelles and even whole cells, if they can be squeezed onto a thin layer, can be directly vitrified in that way.14 Most eukaryotic cells, however, and all tissues are in a larger range of dimensions. Vitrification at least in the 100 µm scale is needed.


Vitreous Water

11

The cooling speeds calculated above are theoretical maxima. They cannot be significantly increased because the limiting factor is the thermal conductivity of water. There are, however, two other parameters that can favour vitrification: the addition of a cryoprotectant and high pressure. The former acts by reducing the capability of water molecules to participate in crystal formation. For example, in a saturated sugar solution, water molecules are so involved in their interaction with the sugar that they cannot crystallize. Most soluble substances act as a cryoprotectant. The material inside a cell, the cytoplasm, which typically corresponds to 15 to 30 percent dry weight, is a cryoprotectant that nature provides freely. We take advantage of it. High pressure is not so easily offered and its advantage must be paid for.15,16 The method stems from the fact that water increases its volume upon freezing; therefore, great pressure applied during cooling makes ice formation more difficult. The trouble is that pressure has only a minor influence on the volume of water, and considerable pressure must be applied in order to induce a significant effect. 2000 atmosphere is the optimal pressure; it is more than that found in the deepest recesses of the ocean. Applying such a pressure in milliseconds while the specimen is cooled at maximal speed is an engineering challenge that may have some similarity with gun technology. Furthermore, it raises the question as to whether this sudden high pressure may induce structural changes. Certainly it does. The very small compressibility of biological matter suggests, however, that the changes are small; this has been confirmed experimentally. For example, it is surprisingly difficult to kill cells by applying high pressure for a short period of time,17 and reports on structural changes produced rapidly by high-pressure freezing concern quite special systems or very minor transformations.18 Certainly, one should pay careful attention to this problem; however, my personal guess is that the effect of cooling has more important structural consequences than high pressure. There are perhaps other means of helping to achieve vitrification. There are substances that are said to be especially efficient cryoprotectants. Trehalose is one of them. Nature has invented surprising ways to avoid — or to help — ice crystal formation. There are efficient antifreeze proteins in plants and animals,5 and it has been claimed that some other common proteins have similar properties (G. Prulière, personal communication). People have also considered using high frequency electromagnetic waves to prevent ice nucleation. These possibilities are certainly worth creative and careful investigation. Summarizing, we note that vitrification of pure water or of any diluted aqueous solution at normal pressure is easy for thicknesses compatible with direct observation in the electron microscope (µm range). This can be increased by a factor of ten by taking advantage of the natural cryoprotective capabilities of most biological specimens. Another factor of ten is gained by resorting to high pressure.19 The 100 µm range for practical bulk vitrification thus is within reach. Practicalities are described in Chapters 5 and 6.


12


5. OBSERVED RESULTS  Figure on the Chapter’s title page General view: The three forms of ice.

 The three forms of solid water frequently encountered in electron microscopy and their electron diffractogram. At the left is a single, large, ramified hexagonal ice crystal grown from a thin layer of water on a supporting film. At upper right is a layer of small cubic ice crystals grown from a thin vitreous layer rewarmed at 135°C. The electron diffractogram formed of concentric rings is characteristic of the large number of small crystals in the observed field. Note also the dark regions in the two forms of ice; they are Bragg reflexions taking place where the crystal has exactly the correct orientation. At the bottom is a layer of amorphous water formed by low pressure vapour deposition on a cold supporting film. (From Dubochet et al. (1988),4 with permission.) 

 Figure 1.1 The water molecule.

 Water molecules and their arrangement in space. Centred on the oxygen atom, with tetragonal symmetry, two strong covalent bonds attach hydrogen atoms at short distance (0.1 nm), whereas two weaker hydrogen bonds are stretched at longer distance (0.2 nm) toward other oxygen atoms. (A) In a plane, a circle can have up to 6 adjacent neighbours. (B) A water molecule and its 4 bonds. (C) A pair of water molecules attached as in hexagonal ice. The shadowed plane marks 3 coplanar bonds.

 Figure 1.2 Phase diagram of water.

 Phase diagram of water. For an explanation, see Section 3.

 Figure 1.3 Free energy and spinodal.

 Stability of liquid (L) and vapour (V) water close to the boiling point. (A) The liquid is the most stable state (below 100°C). (B) The vapour is the most stable state (above 100°C). (C) After the spinodal line, the liquid state exists no longer.




Vitreous Water

13

Figure 1.1

Figure 1.2

Figure 1.3


14


6. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Eisenberg, D. and Kauzmann, W. The Structure and Properties of Water, Oxford University Press, Oxford, 1969. Franks, F. Properties of aqueous solutions at subzero temperatures, in Water: A comprehensive treatise. Water and Aqueous Solutions at Subzero Temperatures, Franks, F. ed., Plenum Press, New York, 1982, 215. Debenedetti, P.G. Metastable Liquids, Princeton University Press, Princeton, 1996. Dubochet, J. et al. Cryo-electron microscopy of vitrified specimens, Q. Rev. Biophys., 21, 129, 1988. Ball, P. H2O; A Biography of Water, Weidenfeld & Nicolson, London, 1999. Angell, C. A. Amorphous water, Ann. Rev. Phys. Chem., 55, 559, 2004. Luyet, B.J. and Gehenio, P.M. Life and Death at Low Temperature, Biodynamica, Normandy, MO, 1940. Mayer, E. and Brüggeller, P. Complete vitrification in pure liquid water and dilute aqueous solutions, Nature, 288, 569, 1980. Dubochet, J. and McDowall, A.W. Vitrification of pure water for electron microscopy, J. Microsc., 124, RP3-RP4, 1981. Mishima, O., Calvert, L.D., and Whalley, E. An apparently first-order transition between two amorphous phases of ice induced by pressure, Nature, 314, 76, 1985. Al-Amoudi, A., Dubochet, J., and Studer, D. Amorphous solid water produced by cryosectioning of crystalline ice at 113K, J. Microsc., 207, 146, 2002. Dubochet, J. et al., Freezing; facts and hypothesis, Scanning Microsc. Suppl., 5, S11— S16, 1991. Studer, D. et al. Vitrification of articular cartilage by high pressure freezing, J. Microsc., 179, 321, 1995. Garvalov, B.K. et al. Luminal particles within cellular microtubules, J. Cell Biol., 174 (6), 759, 2006. Moor, H. Theory and practice of high pressure freezing, Cryotechniques in Biological Electron Microscopy, Steinbrecht, R.A. and Zierold, K. Springer, eds., Heidelberg, Germany, 175, 1987. Studer, D., Michel, M., and Müller, M., High pressure freezing comes of age, Scanning Microsc. 3, 253, 1989. Sato, M. et al. Schizosaccharomyces pombe is more sensitive to pressure stress than Saccharomyces cerevisiae, Cell Struct. Funct., 21, 167, 1996. Leforestier, A. and Livolant, F. Cholesteric liquid crystalline DNA: A comparative analysis of cryofixation methods, Biol. Cell, 71, 115, 1991. Sartori, N., Richter, K., and Dubochet, J. Vitrification depth can be increased more than 10 fold by high pressure freezing, J. Microsc., 172, 61, 1993.


Part I Cryo-Fixation Methods



Slam-Freezing, Metal-Mirror Freezing

19

CONTENTS

GENERAL INTRODUCTION ...................................................................................... 21 1.

PRINCIPLES OF SLAM-FREEZING .............................................................. 22

2.

SUMMARY OF THE DIFFERENT STEPS ..................................................... 23

3.

MATERIALS/PRODUCTS/SOLUTIONS ........................................................ 23 3.1. 3.2. 3.3.

4.

PROTOCOLS ...................................................................................................... 26 4.1. 4.2. 4.3. 4.4. 4.5. 4.6.

5.

Preparation of the Copper Block ................................................................ 26 Preparation of the Slammer ........................................................................ 28 Sample Preparation..................................................................................... 29 Cryo-Immobilization Procedure ................................................................. 30 Checking the Quality of Freezing............................................................... 34 Further Processing ...................................................................................... 35

ADVANTAGES/DISADVANTAGES................................................................ 39 5.1. 5.2.

6.

Materials ..................................................................................................... 23 Products ...................................................................................................... 26 Solutions ..................................................................................................... 26

Advantages ................................................................................................. 39 Disadvantages............................................................................................. 40

WHY AND WHEN TO USE A SPECIFIC METHOD .................................... 41 6.1. 6.2. 6.3.

For Ultrastructural and Immunolabeling Experiments ............................... 41 To Study Dynamic Events .......................................................................... 41 At Liquid Helium Temperature .................................................................. 41

7.

OBSERVED RESULTS ...................................................................................... 42

8.

REFERENCES .................................................................................................... 46





21

GENERAL INTRODUCTION Visualization inside a cell, a tissue or a whole organism is a fascinating challenge that has exploded in the last century with the development of new microscopes at the light microscopy or electron microscopy level. Light microscopy and, in particular, confocal microscopy has its own limits due to the resolution (see Chapter 21). Electron microscopy aims at acquiring ultrastructural information, which also implies ultraresolution, and therefore, requires the best preservation of samples. This goal was first achieved by molecular structural biologists who preserve their samples in the native state and keep them at low temperature in the cryo-electron microscope. However, only a few organisms, such as viruses or bacteria, can be observed directly in their native state using the bare grids method developed by J. Dubochet and M. Adrian (see Chapters 3, 7 and 9). It has been noted by Bellare et al.3 that the environment (temperature, humidity and chemical atmosphere) is essential for arresting the sample in its native state, and a method was devised to achieve the appropriate conditions. This method was improved for biological applications by P. Frederik,9 who has constructed a robot to plunge samples in a humidity, and temperature-controlled atmosphere (see Chapter 4). Despite the fact that electron microscopes are increasingly more powerful and that, for example, in theory, the electron beam of a FEG 300 kV can go through a 1 µm thick specimen, it is not possible to observe an entire cell or tissue. The largest organism that can be observed in a frozen hydrated state are human platelets.10 Therefore, most biological samples that one wants to observe in an electron microscope have to be sectioned after cryoimmobilization. Sections can be cut directly after cryo-immobilization (see Chapter 11) or after freeze-substitution or freeze-drying and resin embedding (see Chapters 13 and 15). Each method has its advantages and drawbacks, which are described in the chapters mentioned. The initial step of rapidly cooling to cryo-immobilize a sample is probably the most critical one It is amazing to read that freezing techniques appeared very early, just before the 20th century, as the best methods for structural preservation.1 However, “conventional microscopy” involving chemical fixatives and dehydration of the specimen, thus inducing various artifacts in samples, had more success than cryo-techniques. This is perhaps due to the fact that cryo-techniques are difficult to work with and require specific skills, experience and expensive apparatuses. Today, despite the fact that the apparatuses are very expensive, cryo-techniques have gained in popularity due to the development of new techniques, such as cryo-electron tomography (see Chapters 12 and 24). Cryo-immobilization can be carried out on a simple impact freezing machine (a very powerful machine unfortunately no longer on the market is Escaig’s slammer7), on a double-jet freezing machine18 or by high-pressure freezing (see Chapters 5 and 6). This chapter will concentrate on the slamming method also referred to as impact or metal mirror freezing. For electron microscopy this method, which was originally described by van Harreveld and Crowell,27 has been modified and improved by Heuser et al.,12 Escaig,8 Heath,11 and Phillips and Boyne.19 Despite the fact that the method is very easy to perform and the apparatus is inexpensive, very few scientists use it. “The easiest way is from time to time the most difficult way.” So, some tips and tricks included in this chapter can make the slamming method an attractive technique for many applications.


22


1. PRINCIPLES OF SLAM-FREEZING Slamming, the process of rapidly projecting cells or tissue onto a cooled metal block is one of the fastest heat transfer methods. One can expect to get several micrometers in depth of well-frozen material.

 Structural preservation of a 1030 µm thick region of the cell pellet or tissue.7

Principle of the method:23 A solid copper block is cooled in a Dewar flask with liquid nitrogen (LN2). Then, using an insulated manipulator, the copper block is lifted just far enough above the LN2 level that it still remains in the cold atmosphere of evaporated LN2, thus avoiding frosting.

 The metal mirror (MM80 from Leica) is described in this chapter.

 It is possible to slam large tissue fragments of up to 2 cm in diameter (see Chapter 20).

 Other apparatuses exist, however most of them are no longer on the market.

Cells or tissue have to be prepared so that  This step influences the quality of they remain in the best physiological state ultrastructural preservation before slamming:  Cells have to be carefully pelleted as is commonly carried out when harvesting cultured cells.  Tissue has to be excised and kept in an appropriate physiological buffer. 

 It is recommended to cut 0.5 to 2.0 mm thick slices using the tissue slicer developed by Sitte et al.23 and previously described by Stadie and Riggs25 included in the MM80 and CPC package (Leica).

Cells or tissue are rapidly frozen on the  metal mirror block.  Further procedures:  Freeze-substitution (FS)

 See Chapter 13

 Freeze-drying (FD)

 See Chapter 15

 Cryo-sectioning (CEMOVIS)

 See Chapter 11

 Room temperature sectioning after FS or FD


23


2. SUMMARY OF THE DIFFERENT STEPS 1. Preparation of the copper block 2. Preparation of the apparatus 3. Sample preparation

 LN2 is required.  For safety procedures using liquid nitrogen, see Sitte et al.24

4. Slamming procedure 5. Checking vitrification 6. Further processing

 Cryo-ultramicrotomy: CEMOVIS (see Chapter 11).  Freeze-substitution (see Chapter 13).  Freeze-drying (see Chapter 15).  Room temperature sectioning after freeze-substitution or freeze-drying.

3. MATERIALS/PRODUCTS/SOLUTIONS 3.1. Materials  Impact freezer

 Homemade.23  Some are “precious oldies” like the helium Cryovacublock (Escaig) previously sold by Reichert-Jung, Vienna, Austria, and still used in some laboratories.15  CPC (with an automatic LN2 refilling system and microprocessor controlled) from Leica, Microsystems, Vienna.  This chapter will focus on use of the MM80 from Leica (Figure 2.1).

Figure 2.1 MM80 apparatus


24


 Plastic sheets  Plastic cups

  Very dense polystyrene sheet





 Spacer rings

 Tweezers

 Insulated cryo-tweezers


 Orange plastic spacer of a Leitz binder 0.3 mm thick. Small squares of about 3 mm × 3 mm are cut.  Homemade. A 2.4 mm diameter indentation can be made with a punching device, which was formerly used to make EM grids.  The borders of the depression have to be sharp (see Figure 2.2).  Be careful: A belt punching device is not applicable because the rim (A) will be rounded. Consequently, the liquid droplet does not form a meniscus, but flows over and will not be frozen properly.

Figure 2.2 Image and sketch of the plastic cup. The rim (A) has to be very sharp. The liquid droplet (1.5 µL or less) stays in the depression and forms a meniscus.   2 mm thick.  Readily available in any laboratory or can be purchased in a hobby shop.   #16860180; furnished with the impact freezer machine.  For better results, fix the rings on double-sided tape attached on a piece of polystyrene. The best results are obtained with 0.3 mm thick rings.  Those commonly used for EM work, e.g., flat, short and antimagnetic (#5 Dumont & Fils, Switzerland).   The orange ones furnished with the machine. (#16701955) Leica Microsystems, Vienna, Austria.


 Heating plate or oven at 50°C

25  Useful to rapidly dry the tweezers, copper block and plastic cups before each slam.

Figure 2.3 A metal block (arrow) on filter paper placed on a heating plate, after slamming. Ice from condensed water vapor is melted and evaporated. The two holes visible on the side of the metal block are useful to insert cryo-tweezers and carry the metal mirror.  Precision micropipette with very thin  Preferably the white Eppendorf 110 µL, #0030 073.363, Eppendorf AG, adapted tips 2233 Hamburg, Germany.  Binocular microscope  Cold light source.  Specific material supplied with any of  Refer to the different supplier’s manuals. the apparatus available will not be described here:  Spacer rings  #16860180 Leica.  Specimen holder  #16701887 Leica.  Liquid nitrogen Dewar

 Styrofoam boxes  Cryo-ultramicrotome  Freeze-substitution apparatus  Freeze-drying apparatus

 Room temperature ultramicrotome


 To refill the slamming apparatus.  It is furnished with the CPC from Leica. In the CPC, the temperature is microprocessor controlled and the LN2 refills automatically.  To transfer the specimen after slamming to one of the additional processing machines.  For cryo-sectioning with an adapted holder that clamps flat specimens, # 16701880 Leica.  For freeze-substitution and lowtemperature resin embedding.  For freeze-drying and low-temperature resin embedding.  Cryosorption freeze-drying system (CFD), Leica.

26


3.2. Products  Venol™

    

Liquid nitrogen Cotton hydrophilic wad For cryo-ultramicrotomy For freeze-substitution For freeze-drying

 To clean and polish the copper block before use.  Any other copper cleaning product can be tested and used.   See Chapter 11 and A. Leforestier et al.15  See Chapter 13.  See Chapter 15.

3.3. Solutions  Fetal calf serum  Water-free acetone    Methanol  Epon

 Or any other concentrated serum protein.  Any supplier, e.g., #00570; Fluka, Sigma-Aldrich Chemie GmbH, CH-9471 Buchs SG, Switzerland.  To rinse the copper block after polishing.  M1775GA, Sigma-Aldrich: Fluka, Buchs, Switzerland.  LX112 Embedding kit, Ref. 5.21210, Inland Europe.

4. PROTOCOLS 4.1. Preparation of the Copper Block  Polish the copper block with Venol Use a cotton hydrophilic wad (no paper napkins or other products, such as Kimwipes)


 This step is absolutely necessary.  Paper napkins and Kimwipes slightly scratch the copper block. The striations will interfere with the slamming process after a while.  Make sure that all the Venol is removed before using the copper block. Any trace of the cleaning agent can interfere with the specimen.  It is not necessary to polish the copper block after each freezing run, but only when the sample has scarred the copper block. In real life, this often happens and the copper block has to be cleaned when this occurs.

27


 An alternative to the polishing step is to use gold-covered copper blocks that do not need to be polished after each slam. It just needs washing with acetone or ethanol before each slamming. Figure 2.4 Copper block.  Rinse the block with acetone or ethanol.

Figure 2.5 Copper block held with the orange tweezers.

 Place the copper block in the apparatus and let it cool.

Figure 2.6 The copper block is placed in the apparatus. The two holes on the side are made to pick up the copper block with the orange tweezers to avoid scratching the surface of the metal mirror.  To test if the block has reached the working temperature and is ready for slamming, fill the chamber with liquid nitrogen up to the brass plate (see Figure 2.6, arrow). Cool down the orange tweezers by dipping them in the chamber and remove a drop of liquid nitrogen (only one drop!) and place it rapidly on the copper block.


 If the copper block is cold enough, the drop stays on the copper for a few moments before evaporating.  Has to be done just before slamming.  Wipe all traces of liquid nitrogen off the copper block (when it is cold!) using a precooled, dry cotton wad.  When using the CPC (Leica) instead of the MM80, the temperature can be set and controlled automatically.

28


4.2. Preparation of the Slammer  Fill the MM80 with liquid nitrogen:

 10 to 15 minutes are needed for the apparatus to sufficiently cool down.  When the system has stabilized, care must be taken to avoid pouring liquid nitrogen on the copper or gold-plated copper block.

 Here the metal mirror freezer MM80 from Leica will be described.  In this case, the whole procedure has to be started again: to heat, polish, pour liquid nitrogen, etc.  A beep signal is emitted when the apparatus needs to be filled with liquid nitrogen. When the system has stabilized, i.e., beeping does not occur every couple of minutes, the apparatus is ready to start.  If this happens, cryo-immobilization is not efficient. Under these circumstances, the warm specimen is projected toward the liquid nitrogen covering the copper block. The liquid nitrogen evaporates and produces an insulated gas layer, which prevents cryo-fixation.11  After the light has been switched on, the TF (TransFer of nitrogen vapor) process is activated and will automatically start when the slider is opened. When the TF process is on, LN2 is evaporated and the cold nitrogen vapor fills the chamber to keep the interior dry and at 196°C. This process is essential to prevent the sample from warming up and subsequent ice crystal formation.  Pressing the TF button will cause LN2 vapor formation even if the slider is closed and the lights are off. Figure 2.7 Control buttons of the MM80 (in color on the apparatus)  On the right:  Mains on button: green lamp  Mains off button: red lamp  On the left:  White button corresponds to the light and the TF  Orange button corresponds to the heating mode.

 Settings for the plastic cups:  Thickness = 0  Force = 6  Speed = 6


 Other sample holders can be used and are referred to in the material section.  Thickness: Has to be adapted depending on the specimen holder; a simple way to adapt it is to measure the overall distance of the mounted specimen holder squeezed between the thumb and the index.


29  Force: Choose the same value as speed.  Speed: The best results are obtained with short specimen contact ( 20 nm gold give very low labeling intensity.  Wash  Wash buffer 3 × 10 min  Wash  Double distilled water


1 × 3 min  In solution without BSA, the tissue side of replicas tends to stick onto glassware and platinum wire leading to damaged replicas. Handle them on the nontissue side.

SDS-Digested Freeze-Fracture Replica Labeling (SDS-FRL)

581

 Mounting replica membranes onto the  Replicas should be kept on the water surface and be unfolded with platinum wire EM grids under a stereo microscope.  At this step it is important to have large, intact replica membranes in which information about tissue architecture can be retrieved.  Practice is required in handling replicas in solution. 6. LM observation  Stereomicroscopy

7. EM observation

 Stereomicroscopic images of replicas with transparent illumination and/or reflecting illumination are useful to obtain information about tissue architecture in the replicas.  For investigation of immunoreactivity and morphology of plasma membrane faces.  For interpretation of images, please refer to original papers listed in the reference section.

5. ADVANTAGES/DISADVANTAGES 5.1. Advantages  Two-dimensional visualization of  Instead of reconstructing serial ultrathin protein localization in the plasma sections, SDS-FRL can directly visualize membrane at EM level two-dimensional distribution of membrane molecules. Localization of the immunogold particles coincident with Pt/C replica images of membrane faces are obtained by TEM.   Quantitative analysis of protein  SDS treatment dissociates proteins from localization target proteins in the protein complex and denatures their higher-order structure, resulting in an equal detectability by specific antibodies. Visualization of immunoreactivity with antibodies coupled to colloidal gold particles enables quantitative analysis of protein localization.


582


 Highly sensitive detection and clear-  In the conventional pre-embedding cut distinction of the pre- or post- immunolabeling method, molecules in synaptically localized molecules synaptic sites are often undetectable because of poor penetration of antibodies into the densely packed protein matrix of the specialized synaptic membranes. On the other hand, in the post-embedding immunolabeling method, the deviation of immunogold particles (up to 30 nm) from the antigen site makes it difficult to distinguish pre- and post-synaptic location.   Wide applicability of specific  SDS-FRL uses similar antigen–antibody antibodies reaction conditions to those used for the Western blot immunodetection. Therefore, a large proportion of antibodies usable in the latter method may also be applicable to this technique.

5.2. Disadvantages  Difficult to identify cellular or sub-  Information about fine structure in the cellular origin of each replicated vicinity of replicated membranes is lost. membrane  Difficult to produce replicas com-  Fracture occurs in a random fashion prising specific cellular components within the frozen tissues. within a frozen tissue.  

6. WHY AND WHEN TO USE A SPECIFIC METHOD 6.1. Preparation of Tissue 6.1.1. Fixation



 0.5% FA + 15% saturated PA in 0.1 M  For more synaptic profiles in the replica PB membrane  4.0% FA + 15% saturated PA in 0.1 M  For finer structure of the plasma PB membrane and intramembrane particles (IMPs)



583

6.1.2. Unfixed tissue  Artificial cerebrospinal fluid in which  For some target molecules that show low sodium chloride is substituted with immunoreactivity in the fixed tissue. 0.8% sucrose.   

6.2. Replication 6.2.1. Fracturing and shadowing temperature  Fracture and replication at 130°C or  For finer structure of the plasma membrane and IMPs. below. 

 6.2.2. Shadowing  Rotary shadow for the second layer, 2.5 nm platinum/carbon from 25° with sample rotation. 

 For finer structure of the IMPs.   

6.2.3. Grid-mapping of the replicated tissue  Attach an EM grid to an entire replica  To prevent replica membranes from membrane with Lexan resin before breaking into small pieces during tissue tissue removal. removal and immunolabeling procedures.  For detailed mapping of the cytoarchitecture within replicas. Detailed procedure is described in References 3,5.

6.3. Tissue Removal  Incubations  105°C for 10 min by autoclave

 60°C for one day and 37°C overnight


 When the hybridization oven is not available.   For detection of membrane-associated proteins.3

584


7. OBSERVED RESULTS  Figure on Chapter’s title page

 See Figure 22.7.

 Figure 22.7 1. Description

 Co-localization of AMPA- and NMDAtype glutamate receptors in the postsynaptic specializations in the plasma membrane of the dentate gyrus granule cell. The postsynaptic specialization was identified by a cluster of IMPs on the exoplasmic fracture face.

2. Method

 SDS-FRL

3. Tissue

 Adult rat hippocampus

4. Fixation

 2% FA, 15% saturated PA in 0.1 M PB

5. Fracture temperature

 120°C

6. Coating layers

 5 nm C, 2 nm Pt/C from 60°, 15 nm C

7. SDS treatment

 80°C for 18 hours

8. Antibodies

 Primary antibodies:  Rabbit anti-GluR1-4 antibody was kindly donated by Prof. E. Molnar at University of Bristol U.K. (3.3 g/L).  Mouse anti-NMDA receptor 1 antibody (Chemicon, 3.6 g/L).  Secondary antibodies:  Antirabbit and antimouse IgG coupled with 5 and 10 nm colloidal gold particles, respectively (0.67 g/mL)

9. Bar


 200 nm



585

586


8. REFERENCES

1. 2.

3. 4. 5.

Fujimoto, K. Freeze-fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins. J. Cell Science, 108, 3443, 1995. Fujimoto, K. SDS-digested freeze-fracture replica labeling electron microscopy to study the two-dimensional distributioin of integral membrane proteins and phospholipids in biomembranes: Practical procedure, interpretation and application. Histochem. Cell Biol., 107, 87, 1997. Hagiwara, A. et al. Differential distribution of release-related proteins in the hippocampal CA3 area as revealed by freeze-fracture replica labeling. J. Comp. Neurol., 489, 195, 2005. Tanaka, J. et al. Number and density of AMPA receptors in single synapses in immature cerebellum. J. Neurosci., 25, 799, 2005. Rash, J.E. et al. Grid-mapped freeze fracture: Correlative confocal laser scanning microscopy and freeze-fracture electron microscopy of preselected cells in tissue slices, in Rapid Freezing, Freeze Fracture, and Deep Etching, Severs, N.J. and Shotton, D.M., eds., Wiley-Liss, New York, 1995, 127.



Immunolabeling of Ultra-thin Sections with Enlarged 1 nm Gold or QDots

589

CONTENTS GENERAL INTRODUCTION .................................................................................... 591 1.

PRINCIPLES OF THE METHOD .................................................................. 592 1.1.

1.2. 1.3.

Small Markers .......................................................................................... 592 1.1.1. Small colloidal gold markers ...................................................... 592 1.1.2. NANOGOLD markers ................................................................ 593 1.1.3. Quantum dot (QD) markers ........................................................ 593 Silver Enhancement.................................................................................. 594 Gold Enhancement ................................................................................... 595

2.

SUMMARY OF THE DIFFERENT STEPS ................................................... 596

3.

MATERIALS/PRODUCTS/SOLUTIONS ...................................................... 597 3.1. 3.2. 3.3.

4.

PROTOCOL ...................................................................................................... 602 4.1. 4.2. 4.3. 4.4.

5.

Labeling with Small Markers ................................................................... 602 Appendix 1: Repeated Enhancement/Enhancement after Staining........... 605 Appendix 2: Double Labeling .................................................................. 606 Appendix 3: Inactivation and Blocking Solutions .................................... 606

ADVANTAGES/DISADVANTAGES.............................................................. 608 5.1. 5.2. 5.3. 5.4.

6.

Materials ................................................................................................... 597 Products .................................................................................................... 598 Solutions ................................................................................................... 599

Advantages of Small Markers .................................................................. 608 Disadvantages of Small Markers .............................................................. 608 Advantages of Enhancement Procedures.................................................. 609 Disadvantages of Enhancement Procedures ............................................. 609

WHY AND WHEN TO USE A SPECIFIC METHOD .................................. 610 6.1.

6.2.

Small Gold and QD Markers .................................................................... 610 6.1.1. NANOGOLD.............................................................................. 611 6.1.2. Ultra-small colloidal gold ........................................................... 611 6.1.3. Small QDs................................................................................... 611 Silver Enhancement Techniques............................................................... 611 6.2.1. HQ SILVER................................................................................ 611 6.2.2. R-GENT SE-EM......................................................................... 612 6.2.3. Danscher solution ....................................................................... 612


590


6.3.

6.4.

Gold Enhancement Techniques................................................................ 612 6.3.1. GoldEnhance-EM ....................................................................... 612 6.3.2. Published recipes ........................................................................ 612 Silver Stabilization/Gold Toning.............................................................. 612

7.

OBSERVED RESULTS.................................................................................... 613

8.

REFERENCES .................................................................................................. 615





591

GENERAL INTRODUCTION Very small (1 nm) gold markers, such as ultra-small (colloidal) gold (Aurion) and the gold compound NANOGOLD™ (Nanoprobes), have become more important in electron microscopic immunocytochemistry due to considerably improved sensitivity (see Figure on Chapter’s title page).1,11,22 The main reasons for improved sensitivity are less steric hindrance and reduced electrostatic repulsion (the colloidal gold surface is negatively charged). These markers penetrate into permeabilized cells and tissues more readily than larger gold colloids and, in ultrathin resin and cryo-section labeling experiments, the density of the label is often considerably higher when compared to colloid sizes of 4 nm or larger. In contrast to permeabilized samples, ultrathin resin sections cannot be penetrated by gold markers whatever the marker size used20 (see Figure 23.1). Also in the case of well preserved thawed cryo-sections obtained according to Tokuyasu,25 gold labeling is restricted mainly to the section’s surface, but (local) low matrix density, either due to inherent specimen properties or section damage, may allow some penetration, especially of 1 nm gold markers24 (see Figure 23.2). Due to their small size, the electron density of 1 nm gold particles is low and their visualization in a conventional transmission electron microscope (TEM) is difficult.22 This also holds true for small semiconductor nanocrystals, so-called quantum dot (QDs, e.g., QDOT® (Invitrogen) markers that exhibit low intrinsic contrast.6 Therefore, these markers have to be enlarged, either by deposition of metallic silver (so-called silver enhancement1,4,5,21,22) or gold (so-called gold enhancement26). Both enhancement techniques can be a source of problems, e.g., due to the low stability of the silver layer.1,18,21,22 The aim of this chapter is to help to choose the most useful small marker and enhancement method for a specific application and to cope with the typical practical problems associated with the use of very small markers and the application of enhancement techniques.

Figure 23.1 Cross-section through a gold labeled ultrathin resin section. Due to the high matrix density (polymerized resin, hatched area) luminal (white stars) and cytoplasmic (black) epitopes of vacuolar membrane antigens are only accessible for immunoreagents at the resin section surface (spheres, gold marker). Antibodies are not visualized.


592


Figure 23.2 Cross-sections through goldlabeled, ultrathin Tokuyasu cryo-sections.  (a) In the case of well-preserved sections, gold markers are preferentially bound to the cryosection surface. Section damage (arrow) and local low matrix density in the vacuolar lumen allow some penetration of immunoreagents into the thawed cryo-section.  (b) In the case of weakly fixed and, therefore, extracted sections, antibodies and especially 1 nm gold markers can permeate the thawed cryo-section to a certain extent due to its low matrix density.

1. PRINCIPLES OF THE METHOD 1.1. Small Markers The preparation of 1 nm gold markers and their conjugation to ligands such as antibodies, are considerably more difficult when compared to larger ones.1 Therefore, commercial products are preferred.

 For example, unconjugated 1 nm gold colloids are less stable than larger ones.  Several recipes (for review see Reference 1). 

1.1.1. Small colloidal gold markers The smallest gold colloids in use have sizes between 0.8 nm and ~ 3 nm.1,14,17,21-23 The colloid surface is hydrophobic and negatively charged (due to adsorbed ions). The actual particle size is larger due to an additional hydration shell. Antibodies or antibody fragments are noncovalently (but tightly) bound via hydrophobic and electrostatic interactions. 

 Few commercial suppliers (Aurion (Ultra-small); British Biocell International (2 nm gold); Amersham/GE Healthcare (AuroProbe One).  Relatively large size distribution, e.g., ultra-small gold (Aurion).  Gold colloids have a rigid and thick coat of water dipoles due to their negative surface charge.

Figure 23.3 Fab (antibody) fragment noncovalently bound to 1 nm colloidal gold (black). The Fab fragment is 5 to 6 nm in length. Antigen-binding site (bright ends), IgGs may have more than one colloid disulphide bridge (white rectangle) (drawn to scale). bound.



593

1.1.2. NANOGOLD markers NANOGOLD (Nanoprobes) is a gold compound with a gold core diameter of 1 to 1.4 nm and including the organic shell, 2.7 nm.10,11 Antibodies or antibody fragments are covalently linked to the organic shell via a hinge region thiol (one gold particle per Fab fragment or IgG). 

 One commercial supplier.  There are also double labeled NANOGOLD-antibody (fragment) conjugates, which are additionally linked to fluorochromes like fluorescein or Alexa dyes 488 and 594.  Figure 23.4 Fab fragment covalently linked to the organic shell (grey) of NANOGOLD (gold core, black). Antigen-binding site (bright ends), disulphide bridges (white rectangles) (drawn to scale).

Undecagold (Nanoprobes) is even smaller (gold core 0.8 nm, organic shell 2 nm in  Undecagold can be used to label-specific diameter),11 but cannot be satisfactorily sites on a protein for single particle enhanced by silver deposition. analysis.

1.1.3. Quantum dot (QD) markers QDs are fluorescent semiconductor nanocrystals made of substances, such as cadmium selenide as the core, which is surrounded by a core shell. QDs offer unique optical properties like high quantum yield, resistance to photobleaching and size-tunable emission spectra.6 Core and core shell exhibit moderate electron density6 (see Figure 23.9 a-c).




 Several commercial suppliers of antibody-conjugated nanocrystals, e.g., QDOTs (Molecular Probes/Invitrogen), or EviTags (Evident Technologies/Antibodies Inc.).  Important for immunofluorescence microscopy.  The relatively small, green fluorescent QDOT 525 (Molecular Probes) has an electron dense spherical core/core shell of about 3 to 5 nm in diameter. The whole particle is 11 to 14 nm in diameter including the organic layer and bound (Fab`)2 fragments (see Figure 23.9 a-c).  Figure 23.5 Model of QDOT 525 conjugate: The core (black) is surrounded by a core shell (grey) and an organic layer (black ring), which is covalently linked to several (Fab`)2 fragments. Antigen-binding sites (bright ends), disulphide bridges (white rectangles) (drawn to scale).

594


 There are QDs conjugated to secondary antibodies, which may be smaller (e.g., EviTag Adirondack Green), but it is not known whether they can be silver-enhanced or not.

1.2. Silver Enhancement Gold colloids and organo-gold clusters as well as QDs can act as nuclei, comparable to silver nuclei, in photographic development. In the presence of a reducing agent, the gold surface acts as a catalyst for the reduction of silver ions to metallic silver. The metallic silver deposited on the gold surface itself serves as nuclei for further reduction of silver ions to metallic silver, resulting in a growing silver layer.3,4

 Silver enhancement for immunogold electron microscopy was introduced in 1983.15  The enhancer solution contains a silver salt, a reducing agent, a buffer and often the so-called protective colloid gum arabic, which improves the enhancer’s performance: it makes the reaction more efficient and reproducible.1,3,4,21-23 

Figure 23.6 Principle of silver enhancement. The gold surface serves as a catalyst for the reduction of silver ions, e.g., by hydroquinone. Whereas silver enhancement of larger gold particles is relatively unproblematic, it turned out that enhancement of 1 nm gold is associated with a number of problems, such as inefficient enhancement, uneven growth and increased auto-nucleation due to prolonged enhancement time.1,21-23 A general drawback of silver enhancement is the instability of the silver layer caused by oxidation, which requires additional treatment or special storage conditions for protection.1,18


 As a consequence, not all enhancers are suitable for 1 nm gold markers and QDOTs.1,21-23  OsO4 dissolves the silver layer; electron beam and air humidity cause dislocation/loss of silver (see Figure 23.14).


595

We will describe the use of two commercial  Nickel grids should be used because silver enhancers and one published method: nickel does not interfere with silver enhancement, in contrast to copper.  HQ SILVER (Nanoprobes) contains  See Figure 23.10a, b. the protective colloid gum arabic. Its pH is near-neutral, the viscosity is high.  R-GENT SE-EM (Aurion) is a low  See Figure 23.10c. viscosity enhancer with near-neutral pH.  The Danscher method3,4,23 is an example of a published recipe of high efficiency, which is suited for all small markers mentioned. The enhancer contains the protective colloid gum arabic. The pH is low, the viscosity high.

 See Figure 23.11.  Two other published recipes deserve mentioning: The first one,16 a fast-working enhancer, can be used for copper grids. The second one7 causes less structural damage on whole mount samples or Tokuyasu cryosections than the Danscher method, probably due to a near-neutral pH.

1.3. Gold Enhancement In principle, the deposition of gold instead of silver offers potential advantages: it confers stability to the enhanced particle, thereby making it resistant to oxidation.1,18,26

GoldEnhance™-EM (Nanoprobes) is the only commercial product available (Near neutral pH, low viscosity, less sensitive to buffer salts).26 Gold chloride treatment has been described as being able to enlarge small colloidal gold markers (for review see References 1 and 18).


 A process similar to silver enhancement.  Higher particle density (improved contrast).  Improved backscatter detection in the scanning electron microscope (SEM).  Caused by OsO4 treatment, beam damage, and air humidity.   In our lab, gold markers bound to resin sections can be readily enhanced (see Figure 23.12), but treatment of labeled cryo-sections results in strong background.   In our lab, gold enhancement following published procedures leads to loss of gold rather than to gold particle growth1,18 (see Figure 23.13). 

596


2. SUMMARY OF THE DIFFERENT STEPS Ultra(cryo)microtomy and mounting of sections:  Mounting ultrathin cryo-sections (swimming on the surface of the pickup drop; see Chapter 19) or resin sections on plastic- and carboncoated13 grids (Figure 23.7, top and middle).  Transfer of grids (upside down) to drops of buffer placed on a hydrophobic film in a dish covered by a glass plate (moist chamber) (Figure 23.7, middle and bottom).  Optional: Cryo- and resin sections mounted on grids can be stored prior to immunolabeling.8 Figure 23.7 Mounting of sections on grids (top, middle) and transfer of grids to drops of buffer (middle, bottom) for immunolabeling.

Figure 23.8 Labeling of grids in moist chamber. Grids can be transferred with a forceps or a loop (see also Chapter 21). 1: Inactivation of residual fixative molecules (mainly aldehyde groups) (optional) 2: Blocking unspecific protein binding sites 3, 4: Antibody labeling 5, 6: Marker incubation 7, 8: Fixation 10, 11: Silver or gold enhancement 12, 13: Stabilization of silver layer (optional) (gold toning) 14: Staining (heavy metals) 15, 16: Drying (resin sections) or methyl cellulose embedding (cryo-sections)



597

3. MATERIALS/PRODUCTS/SOLUTIONS 3.1. Materials  Vortex  Centrifuge  Forceps (antimagnetic)  Petri dish (glass)  Dish (stainless steel) with glass plate  Hydrophobic film  Loops (stainless steel, 3 mm in diameter) attached, e.g., to pipette tips  Tissue paper/wipes  Pioloform or Formvar coated and carbon-covered nickel grids for silver enhancement, copper or nickel grids for gold enhancement  Pipettes (glass or plastic)  Pipette tips  Desiccator (made of glass or plastic)  Silica gel-desiccant  Black cardboard  Filter paper  Water bottle (containing distilled water)  H2O

double

 Microtubes (0.5 mL, 1.5 mL, 2 mL)


 For antibodies, markers, and enhancers; e.g., Vortex-Genie 2, Scientific Industries Inc, Bohemia, New York., USA.  For all solutions used; e.g., Centrifuge #5415D, Eppendorf-Netheler-Hinz GmbH, Hamburg, Germany.  Grid transfer (nickel grids!); e.g., # 5/45 or #7, Dumont & Fils, Switzerland.  For lead staining.  Moist chamber for labeling and enhancement.  For incubating grids on drops; Parafilm “M”, Pechiney Plastic Packaging, Chicago, USA.  Self-made loops from platinum wire for grid transfer and for drying cryo-sections.  For generating humid air; e.g., Kleenex®, Kimberly-Clark Corp, USA.  For supporting sections (100 to 200 mesh nickel grids), e.g., Stork Veco B.V, Eerbeek, The Netherlands; or Gilder Grids, Grantham, England.  For plastic and carbon covering of grids, see Reference 13.  For placing drops.  For dilution, placing drops, etc. Eppendorf-Netheler-Hinz GmbH, Hamburg, Germany.  For storing silver-enhanced sections in dry air, nitrogen gas, or vacuum.  For dry storage of silver-enhanced sections.  For dark chamber for light sensitive reactions (e.g., silver enhancement).  For drying grids, usually Whatman 541, Whatman International Ltd, Maidstone, England.  For jet wash of grids.  Water, double distilled (on quartz) or Milli Q.  For mixing solutions, for storing frozen aliquots; Eppendorf-Netheler-Hinz GmbH, Hamburg, Germany or Sarstedt AG & Co, Nümbrecht, Germany.

598


3.2. Products  Ammonium hydroxide (NH4OH)                    

         

 For example, Merck Eurolab, VWR International; Darmstadt, Germany. Sigma-Aldrich, Fluka, Buchs, Bovine serum albumin (BSA)  Switzerland. fraction V Bovine serum albumin, acetylated  Aurion, Wageningen, The Netherlands. (BSA-c)  Merck. Citric acid (C6H8O7 x H2O)  Aurion, Sigma. Cold water fish gelatin Disodium hydrogenphosphate  Merck. (Na2HPO4)  Merck. Ethanol  Gibco, Invitrogen, Eugene, Oregon, Fetal / new born calf serum USA.  Merck. Gelatin  Sigma #G-5882 (keep frozen). Glutaraldehyde (aqueous, 25%)  Merck. Glycine  See tetrachloroauric acid trihydrate. Gold chloride  Merck. Gum arabic  Merck. HEPES  Merck. Hydroquinone  Sigma #L-7771 (Caution: light L-(+)-lactic acid sensitive). Lead (II) citrate trihydrate  Fluka, Buchs, Switzerland; or Agar Scientific. (Pb3(C6H5O7)2 x 3H2O)  Serva 29834, Heidelberg, Germany. MES (C6H13NO4S)  Sigma M-6385. Methyl cellulose  Regular non fat milk powder. Milk powder (non fat, dried)  Nanoprobes (Stony Brook,New York, NANOGOLD™ conjugates USA).  Ultra-small gold and NANOGOLD conjugates can be stored at 20°C after mixing equal parts of marker and 99% glycerol.  Invitrogen. Nonimmune serum / normal serum  Merck. N-propyl-gallate Ovalbumin (albumin from egg white)  Sigma.  Merck. Oxalic acid (C2H2O4 x 2H2O)  Merck. Potassium chloride (KCl) Potassium dihydrogen phosphate  Merck. KH2 PO4  Molecular Probes, Invitrogen, Eugene, Quantum dot (QDOT®) conjugates Oregon, USA.  Merck. Sodium borohydride (NaBH4) Sodium chloride (NaCl)  Merck.  Merck. Sodium hydroxide (NaOH) pellets



 Sodium thiosulfate (Na2O3S2)  Tetrachloroauric(III)acid trihydrate (AuCl4H x 3H2O)  Trisodium citrate dehydrate (C6H5Na3O7 x 2H2O)  Tween-20®  Ultra-small gold conjugates  Uranyl acetate (UO2(CH3COO)2 x 2H2O)  Uranyl oxalate

599

 Merck.  Merck #101582.  Merck.  Sigma, Serva.  Aurion.  Stain for electron microscopy. EMS, Fort Washington, Pennsylvania, USA or SPI Supplies, West Chester, Pennsylvania, USA.  See Staining solutions. 

3.3. Solutions 1. R-GENT SE-EM Activator, initiator,  Aurion, Wageningen, The Netherlands.  Shelf life ~ 18 months from date of enhancer production.  Preparation of developer:  Light insensitive.  Put 40 drops of activator (1385 µL) into the developer bottle  The concentrated initiator can be stored  Add 1 (36 µL) drop initiator to the in a freezer. developer bottle  Shelf life of developer is about one  Mix well on a vortex month.  Use nickel grids.  Preparation of final enhancer:  Mix 20 drops (870 µL) of enhancer with 1 drop (34 µL) of developer  Mix well on a vortex 2. HQ SILVER  Moderator, activator, initiator  Equal amounts of moderator, activator, and then initiator are thoroughly mixed before use Danscher (acidic) silver enhancer3,23 Stock solutions: Gum arabic: 33% (w/v) in H2O Hydroquinone: 0.57 g in 10 mL H2O (5.7%, 0.52 M)  Citrate buffer: 2.55 g citric acid (23.5%, 1.5 M) + 2.35 g trisodium citrate dehydrate (23.5%, 0.5 M), add H2O to make 10 mL (pH 3.53.8)  Silver lactate: 7.3 g in 10 mL H2O (0.73 %, 37 mM) 3.   


 Nanoprobes, Stony Brook, New York, USA  Solutions should be kept frozen in small aliquots.  Caution: Light sensitive.  Shelf life ~ 30 months.  Use nickel grids.  Stir for one day, centrifuge (30,000 g, 2 h, 4°C).  Gum arabic, citrate buffer, and hydroquinone can be premixed and stored in a freezer. Silver lactate has to be stored separately in a freezer.  Caution: Light sensitive!  Caution: Silver lactate salt may age with time and change properties. If in doubt, buy a new package.

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 Preparation of final enhancer:  Mix 6 parts gum arabic stock with 1 part citrate buffer stock and 1.5 parts  Use nickel grids. hydroquinone stock. Add 1.5 parts  Caution: Moderate light sensitivity after silver lactate stock and mix mixing. thoroughly. 4. Fast working silver enhancer16  Suitable for copper grids.  See above, replace citrate buffer by  Caution: Light sensitive! 0.2 M HEPES buffer, pH to 6.8 with NaOH.  Reduced ultrastructure damage 5. Neutral silver enhancer7  Stock solutions: (probably due to near-neutral pH).  0.5 M MES adjusted to pH 6.15 with NaOH  Gum arabic: 33% (w/v) in H2O  N-propyl gallate: dissolve 10 mg NPG in 0.25 mL ethanol, then add 4.75 mL of H2O  Silver lactate: 36 mg in 5 mL H2O  Caution: Light sensitive!  Preparation of final enhancer:  2 parts MES stock, 5 parts gum arabic stock, 1.5 parts N-propyl gallate stock,  Use nickel grids. and 1.5 parts silver lactate stock.  The first three components are mixed 30 min before use, the N-propyl  Caution: Light sensitive (dark room)! gallate stock is added 2 min before use. 6. GoldEnhance™-EM  Mix equal amounts of enhancer, activator, initiator, and buffer.  Mix enhancer and activator, wait 5 to 10 min, then add initiator and buffer.

 Nanoprobes, Stony Brook, New York, USA.  Shelf life ~ 36 months.  Use copper or nickel grids.  For numerous modifications, see www.nanoprobes.com.

PBS buffer (pH 7.4)  Standard buffer. 137 mM sodium chloride (NaCl) 2.7 mM potassium chloride (KCl) 8.1 mM disodium hydrogen-phosphate (Na2HPO4)  1.5 mM potassium dihydrogen phosphate (KH2 PO4)

7.   


601


8. Staining solutions  Uranyl oxalate or neutral uranyl acetate:25  2% (w/v) uranyl acetate in 0.15 M oxalic acid, pH 7.4

 Stains for resin and cryo-sections.  Stir a 0.3 M oxalic acid solution vigorously and add slowly the same volume of a 4% (w/v) aqueous uranyl acetate solution. Adjust pH to 7.4 with 10% ammonium hydroxide.25  Store in refrigerator.

 4% (w/v) aqueous uranyl acetate  1% (w/v) aqueous uranyl acetate

 Store in refrigerator.  Store in refrigerator.

9. Methyl cellulose  2% (w/v) methyl cellulose in H2O (25 centipoises).  Methylcellulose–uranyl acetate (UA) solution:  3% UA in H2O  2% aqueous methyl cellulose  Mix 1 (1.5) part UA with 9 (8.5) parts methyl cellulose.

 Embedding of cryo-sections.  Centrifuge at 100,000 g at 4°C.  Store in refrigerator.  See also Chapter 19.  Stronger contrast and darker structures with higher UA concentration. Too high UA concentration causes membrane blebbing.25

10. Lead citrate staining  Recipe

 Stain for resin sections.  See Chapter 21.

11.

 See gold toning.

Gold chloride

12. Gold toning  Protection of silver layer.  0.05% aqueous gold chloride (diluted  Store in darkness (at 4°C). from 1 to 2% stock solution of tetrachloroauric acid trihydrate) 13. Fixative  0.5 to 1% aqueous glutaraldehyde

 Stabilization of antigen-antibody-marker complex prior to enhancement.

14. Blocking buffer  See Appendix 3 for alternative reagents.  0.5% BSA plus 0.5% nonfat milk powder in PBS 15. Glycine-PBS  Inactivation  50 mM glycine in phosphate buffer aldehyde groups. saline (PBS)

of

residual

16. Silver fixer, e.g.,  To stop silver enhancement.16  250 mM sodium thiosulfate in 20 mM HEPES (pH 7.4)


reactive

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4. PROTOCOL 4.1. Labeling with Small Markers Labeling with small markers

 Indirect labeling.  All of the following steps are carried out at room temperature (20 to 22°C) in a moist chamber.  The entire immunolabeling and silver enhancement of sections mounted on grids is carried out on 50 to 100 L drops of solutions placed on a hydrophobic film (in a dish covered by a glass plate) (see Chapter 21).  All solutions should be centrifuged before use (13,000 g/2 to 3 min) to pellet aggregates and debris and to remove bubbles.

Ultrathin resin/cryo-sections collected on  Nickel does not interfere with silver Pioloform and carbon-coated13 nickel grids enhancement, in contrast to copper.  Copper and nickel grids can be used for (e.g., 100 or 200 mesh) gold enhancement.  Formvar or collodium coating is also possible.13  1. Inactivation of residual fixative  Mainly aldehyde groups (see Section molecules 4.3) Optional.  Glycine (50 mM) in 510 min  See Section 4.4. PBS 2. Blocking unspecific protein-binding sites  Blocking buffer 2 × 710 min  See Section 4.4, for different recipes. 0.5% BSA, 0.5% milk powder in PBS 3. Primary antibody incubation   Antibody diluted in 10 µL/grid  Detection of antigens. blocking buffer 3060 min  Final (specific) IgG concentration 1 to 5 µg/mL.  Moist chamber.  For double labeling, see Section 4.2. 4. Wash  Blocking buffer


6 × 35 min  Removal of unbound antibodies.


5. Marker incubation  Marker conjugate diluted in blocking buffer 6. Wash  Blocking buffer  PBS 7. Fixation  0.5% glutaraldehyde in PBS 8. Wash  H2O

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 Detection of bound primary antibodies. 10 µL/grid  NANOGOLD conjugates: 1:50 to 1:100.  Ultra-small gold conjugates 1:20 to 1:50. 3060 min  QDOT conjugates: 1:20.  Moist chamber.  Removal of unbound markers. 2 × 35 min 4 × 35 min  To stabilize antigen-antibody-marker 5 min complexes during subsequent treatment.  To remove ions that could interfere with 5 × 23 min silver enhancement (e.g., chloride).

9. Drying (only resin sections)

 Optional.

10. Silver (a-c) or gold (d) enhancement

 To make small markers visible.  Caution: Speed of enhancement depends on temperature, higher temperatures will speed up deposition of silver or gold.  Enhancement of larger gold is less problematic. All the silver enhancers mentioned as well as GoldEnhance-EM can be used. Caution: Enhancement time is shorter due to larger colloid size.

10a. HQ SILVER  Incubation  NANOGOLD  Ultra-small gold  QDOT 525 10b. R-GENT SE-EM  Incubation  NANOGOLD  Ultra-small gold  QDOT 525 10c. Danscher solution  Incubation  NANOGOLD  Ultra-small gold  QDOT 525


 Three solutions. 50100 µL/grid  Caution: Light sensitive. in darkness 89 min 56 min  (Inefficient enhancement). ~ 5 min  Four solutions. 50100  Light insensitive. µL/grid  Not recommended, no enhancement. 4050 min  Not recommended, inefficient enhancement.  Four solutions (or two solutions when stored frozen).

50100 µL/grid  Moderate light sensitivity. in darkness 3545 min 3035 min  Best enhancer for QDOT 525. 30 min

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10d. GoldEnhance-EM  Incubation  NANOGOLD  Ultra-small gold  QDOT 525 11. Wash  H2O

 Four solutions. 50100 mL/grid  Low light sensitivity. (in darkness) 3060 sec 3060 sec  Not recommended, inefficient enhancement.  On drops, to stop reaction (and remove 5 × 4 min gum arabic).  R-GENT SE-EM: 5 × 2 min is sufficient.  For more rigorous halt of reaction, if desired: fixation with 250 mM sodium thiosulfate, 20 mM HEPES, pH 7.4 (5 min).16

12. Stabilization of silver layer by gold  Optional, to prevent redistribution or toning loss of silver due to oxidation (not necessary for gold enhancement).  0.05% gold chloride 13. Wash  H2O 14.

50 µL/grid  Freshly diluted from stock solution. 15 min  To remove ions that could interfere with 5 × 2 min staining, e.g., phosphate.

Staining

14a. Resin sections  1% aqueous uranyl acetate (UA)  Wash  H2O  Drying  Lead citrate solution, in Petri dish with NaOH pellets  Wash  H2O  Drying


20 µL/grid  See Step 15b. 15 min  Depending on desired contrast. 2 × 5 sec  Final washing in a jet of H2O (wash bottle).  Optional, e.g., to check labeling in TEM. 20 µL/grid 15 sec3 min  Depending on desired contrast.  Keep Petri dish closed to prevent contact with CO2. 2 × 1 sec  Final washing in a jet of H2O from a wash bottle.


14b. Cryo-sections  Uranyl oxalate  Or 1% UA

 Wash  H2O

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20 µL/grid  Optional. 10 min  We have never observed deleterious effects of UA or neutral uranyl oxalate on the silver layer during resin or cryo-section staining or in preembedding labeling experiments when samples were treated with aqueous UA for one hour before dehydration. However, silver-enhanced gold in epoxy sections (if samples were enhanced before embedding) is sensitive to oxidation (electron beam, air humidity). 2 × 1 sec

15. Embedding (only cryo-sections) 50 µL/grid  Numerous recipes for staining and a. Infiltration with 0.3 10 min embedding, see Chapter 19. to 0.5% UA in on ice  Facilitates infiltration with methyl methyl cellulose cellulose. b Picking up grids with a loop and  Dry methyl cellulose film should display removal of excess methyl cellulose by an interference color between gold and touching the loop at an angle of 45° to blue, see Chapter 19. 90°C to a filter paper (section side down). 16.

Air-drying

 In loop; after drying, remove grids carefully (see Chapter 19).

17. Storage (only for nonstabilized silver)  In dry air, under nitrogen gas or under  To prevent redistribution or loss of silver vacuum (in desiccator). due to oxidation.

4.2. Appendix 1: Repeated Enhancement/Enhancement after Staining  Repeated enhancement is possible  Except for the area illuminated by the 21,22 even after inspection in the TEM electron beam. (labeled) Lowicryl sections that were already stained with aqueous UA and  After washing the sections on several Pb citrate can be silver-enhanced. drops of H2O.


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4.3. Appendix 2: Double Labeling 1. Simultaneous or sequential labeling and simultaneous enhancement: After completing the labeling procedure (sequential or simultaneous labeling of two antigens), both markers can be enhanced in one enhancement step.

 Simultaneous labeling: Two specific antibodies, and later two markers are applied simultaneously.  Sequential labeling: In a first round, the first specific antibody is applied, followed by the corresponding marker. In a second round, the second specific antibody is applied, followed by the corresponding marker. A fixation step (e.g., 0.5% glutaraldehyde) after the first labeling round is recommended to stabilize the antigen-antibody-marker complex, provided the “second” antigen tolerates this treatment. Sections should be washed and blocked again before labeling the “second” antigen.  Sequential enhancement: Only possible if the “second” antigen tolerates the enhancement procedure.

2. Sequential labeling and sequential enhancement: Sections can be enhanced twice; after labeling the first antigen and after labeling the second antigen. This means that the first gold marker is silver enhanced twice.

 It is possible to use 1 nm gold markers twice (only for sequential labeling/enhancement19).  Caution: Sizes of silver-enhanced particles are not uniform, so care has to be taken to ensure that there is no overlap in particle size distribution curves of the two enhanced markers.  Caution: Gold chloride treatment results in partial disintegration of the silver layer and, therefore, is not compatible with double labeling or quantification studies.

4.4. Appendix 3: Inactivation and Blocking Solutions Undesirable interactions between antibodies and specimen/resin can be a serious problem. Primary antibodies and secondary antibodies can contain antibodies that recognize epitopes, which differ from the epitope under investigation. Non-specific background can derive from reaction of antibodies with residual fixative molecules, mainly aldehyde groups.


 Such antibodies can be eliminated, e.g., by immunoaffinity purification12 (column, beads, dot blot) or by absorption on specimens lacking the antigen under investigation.


From a theoretical point of view, fixative inactivation should be done directly after completing fixation. Most probably, after resin embedding, but also after cryosectioning, there are no reactive fixative sites left. Nonspecific background can also derive from hydrophobic interactions between sample and antibodies, and from ionic interactions, e.g., from positively charged specimen compounds, such as histone proteins in the nucleus, starch grains in plant cells, or molecules like polycations and collagen. In contrast, negatively charged areas (phospholipids, aldehydefixed proteins) can repulse negatively charged antibodies, thereby reducing label density. In general, blocking should be done not only before but also during antibody and marker incubation because, with time, blocking molecules can also detach from the specimen. For a detailed discussion, see Reference 9 and Web site of Aurion (www.aurion.nl).

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 Useful inactivation strategies: Aldehyde groups can be inactivated by amino acids like glycine or lysine (50 mM) and molecules like ammonium chloride (NH4Cl; 50 mM).

 Useful protein blocking strategies: Gelatin (0.2 to 0.5%), liquid cold water fish skin gelatin (0.5% or 0.005%), ovalbumin (0.5%), bovine serum albumin (BSA) (0.5%), fetal or newborn calf serum (0.5%), nonimmune serum (0.5 to 5%), especially from species used for secondary antibody production), and nonfat dry skim-milk are the most common blocking agents.9 BSA (negatively charged) is often used together with (cold water fish) gelatin or nonfat dry skimmilk powder. In our lab, it turned out that milk powder is the strongest blocking agent. Acetylated (linearized) BSA-c (0.1%, used during antibody and marker incubation) displays increased negative charge and hydrophobicity (www.aurion.nl). It is thought to mimic gold surface properties, therefore, it competes with gold colloids during binding to unspecific sites. Caution: Do not mix BSA-c with gelatin as gelatin exhibits a high tendency (low gold number) to adsorb to gold surfaces. Electrostatic interactions can be also influenced by buffers displaying increased ionic strength, e.g., 0.5 M NaCl or KCl. Hydrophobic interactions can be also influenced by detergents added to the antibody solution, e.g., 0.05% Tween-20, and are also used to reduce background. It should be kept in mind that detergents also extract lipids/cytoplasmic material and enhance wettability (i.e., accessibility of antigens, e.g., in entire mount labeling experiments).


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5. ADVANTAGES/DISADVANTAGES 5.1. Advantages of Small Markers  Often considerably higher label density and efficiency when compared to larger gold markers.  NANOGOLD seems to be the most sensitive gold marker available for labeling resin sections, cryo-sections or permeabilized specimens.  NANOGOLD (with bound fluorochrome) and fluorescent QDs can be used for correlative light/electron microscopy.  NANOGOLD conjugation kits available.  Custom labeling service for colloidal ultra-small gold and NANOGOLD.

 Necessary to detect scarce antigens.  Most probably, fluorochrome-coupled IgGs are even more sensitive (for LM).  Due to less electrostatic repulsion and smaller hydration shell when compared to colloidal gold (see above).  Useful for permeabilized samples, but also for resin and cryo-sections, which can be labeled in parallel for LM and TEM.  Covalent conjugation to thiols or primary amines of antibodies.

5.2. Disadvantages of Small Markers  Low intrinsic contrast, requires additional enhancement step.  Only a few commercial suppliers, quality of conjugate should be checked before use.  Risk of increased background.      

 Enhancement is a source of additional problems.  Use test sample with known label density; check every batch for aggregates, label efficiency, unspecific background.  For example, due to higher sensitivity (see Section 4.3). After resin labeling, unenhanced  Enhancement should be performed NANOGOLD particles are unstable. directly after the immunolabeling procedure. NANOGOLD and Qdots are degraded  Silver or gold enhancement should be by some chemicals and high done directly after immunolabeling. temperatures. No protein A-1 nm gold conjugate.  Due to inactivation of protein A by coupling.  Due to difficulty in obtaining uniformly Quantitation is difficult. sized enhanced particles and unknown stochiometry. Double/multiple labeling is difficult,  Due to difficulty in obtaining uniformly but possible. sized enhanced particles (see Section 4). QDs are relatively large.  Due to the “coating” of the nanocrystal core necessary for stabilization and coupling to antibody (fragments).  Reduced penetration requires strong permeabilization for whole mount labeling.



609

5.3. Advantages of Enhancement Procedures  Small sensitive markers with low intrinsic contrast can be visualized in LM, conventional TEM, STEM, and SEM.21,22

   

 Final marker size can be freely chosen  Suitable for overview and detailed by varying enhancement time. images.   Gold-enhanced particles are stable  In contrast to silver-enhanced particles. against oxidation.  For example, by OsO4, beam damage, air humidity.   Gold chloride treatment (gold toning)  Enhanced gold markers are no longer stabilizes silver-enhanced particles. sensitive to oxidation. 

5.4. Disadvantages of Enhancement Procedures  The use of nickel grids is strongly  Exception: The recipe published by Lah 16 recommended for silver enhancement. et al is compatible with copper grids.   Especially true for the most efficient  Often long enhancement times. enhancers.   Not all commercial enhancers are  See 10, Section 4.1. useful for all markers.  Large size distribution of enlarged  Double (multiple) labeling difficult but possible (see Section 4.2). gold and QDs.  Quantitation difficult (unknown stochiometry).   Silver and gold enhancement products  Quality of enhancer should be checked before use, e.g., by using a test sample can be a source of problems. (sections with known label density); check on silver precipitates (unspecific background), enhancement efficiency, enhancement time.   Silver is sensitive to OsO4 treatment  Requires gold toning before OsO4 treatment, otherwise OsO4 treatment should (oxidation). be reduced in time and concentration (e.g., 0.1%; 15 min) or omitted; or gold has to be “over” enhanced.2


610


 Silver layer is sensitive to oxidation  Requires gold chloride treatment or when sections have been exposed to special storage conditions for long-term electron beam and/or air humidity storage (e.g., in nitrogen gas or in vacuum). (storage) (see Figure 23.12).  Some enhancers are harmful to  Use enhancer with neutral pH or with ultrastructure (especially important for short enhancement time (R-GENT SE-EM, cryo-section, preembedding, and HQ SILVER, or see references 2, 7, 16.). whole mount labeling).2,7,16  Gold enhancement (GoldEnhance-EM) may be problematic with respect to enhancement efficiency, reproducebility, and background. Published enlargement procedures using gold chloride do not work in our lab.1,18

 Use test sample with known label density; check on enhancement time, enhancement efficiency and gold precipitates (unspecific background).  Gold markers become even smaller18 (see Figure 23.13).  The silver layer decomposes in several smaller silver colloids18 (see Figure 23.13).

 Silver-enhanced gold is not compatible  Silver-enhanced gold emits fluorescent with fluorescence markers in light light when illuminated with a Hg lamp.21,22 microscopy.

6. WHY AND WHEN TO USE A SPECIFIC METHOD 6.1. Small Gold and QD Markers  High sensitivity markers should be  Reduced steric hindrance, reduced used for low copy number antigens. electric repulsion, improved penetration properties.  All markers require routine tests for quality.  Useful for resin section, cryo-section,  Limited suitability for double or multiple and preembedding/whole mount label- labeling due to large size distribution. ing.  All markers require additional  Susceptible to enhancement artifacts. enhancement for LM, and conventional TEM and SEM.  Suitable for high resolution studies as  well as for overview images.   



611

6.1.1. NANOGOLD  NANOGOLD is probably the most  For low copy number antigens. sensitive gold marker available.  Not all enhancers work with NANOGOLD.  Best “prerequisites” for penetration  Especially for cryo-section and preinto sample. embedding/whole mount labeling.  Gold/fluorochrome conjugate.  For correlative light/electron microscopy

6.1.2. Ultra-small colloidal gold



 Sensitivity slightly lower compared to  For low copy number antigens. NANOGOLD markers.  Penetration properties slightly poorer  Relevant to cryo-section and prewhen compared to NANOGOLD embedding/whole mount labeling. markers.

6.1.3. Small QDs



 Sensitivity between 4 to 6 nm colloidal  For low copy number antigens. gold and 1 nm gold markers  For cryo-section and resin section labeling, limited suitability for whole mount labeling.  Fluorescent marker of low electron  For correlative light/electron microsdensity, which can be silver-enhanced. copy. 

6.2. Silver Enhancement Techniques  Necessary to visualize small markers  All enhancers require routine tests for in LM, conventional TEM and SEM. quality.  Nickel grids should be used (for TEM).  Suitable for overview and detailed  Caution: Silver is sensitive to oxidation; images. stabilization useful prior to OsO4 treatment and for long-term storage (see Section 6.4). 6.2.1. HQ SILVER  Suitable for all gold and QDOT  Tailored specifically for NANOGOLD  Slightly reduced efficiency for markers and all labeling techniques. QDOT 525.  Low/no interference with ultrastruc-  Important for cryo-section and preembedding/whole mount labeling. ture preservation.


612


6.2.2. R-GENT SE-EM  Suitable for all colloidal gold markers  Tailored specifically for ultra-small gold. (all labeling techniques).  Not suitable for NANOGOLD and QDOT 525.  No interference with ultrastructure  Important for cryo-section and prepreservation. embedding/whole mount labeling.

6.2.3. Danscher solution  Suitable for all gold and QDOT  Easy to prepare. markers and all labeling techniques.  Relatively cheap.  Premixed aliquots can be stored frozen.  May be harmful to ultrastructure in cryosections and preembedding/whole mount labeling experiments.

6.3. Gold Enhancement Techniques  Gold enhancement results in stable  Gold-enhanced particles are not sensitive either to OsO4 treatment, electron beam or gold particles. air humidity.

6.3.1. GoldEnhance-EM  Suitable for all gold markers.

 Limited reproducibility?  Slightly reduced efficiency?  Not suitable for QDOT markers.  Strong background on labeled cryosections.

6.3.2. Published recipes  Not recommended.

 Treatment with gold chloride is not suitable for enlargement of small gold markers.

6.4. Silver Stabilization/Gold Toning  Recommended for stabilization of silver-enhanced gold particles for long term storage and against the action of OsO4.  Alternatively, storage in dry air, under nitrogen gas or vacuum (e.g., in desiccator).


 Gold chloride-treated particles are no longer sensitive to oxidation.  Caution: Gold chloride treatment slightly reduces the size of silver-enhanced particles and leads to their disintegration (see Figure 23.15).


613

7. OBSERVED RESULTS  Centriole labeling in ultrathin cryosections of U2OS cells fixed with 8% FA. C-Nap1 was detected at the proximal end with specific rabbit antibodies and protein A-15 nm gold (left) or silver-enhanced NANOGOLD coupled to goat anti-rabbit F(ab)2 fragments (right). Bar = 250 nm

 Figure on Chapter’s title page

Ultrathin resin sections of Escherichia. coli cells embedded in methacrylate Lowicryl HM20 were incubated with rabbit antibodies raised against the outer membrane protein OmpA. In this overproducing strain, OmpA is mainly found in the periplasmic space. Markers used were protein A-6 nm colloidal gold, ultra-small (colloidal) gold, NANOGOLD, or QDOT 525. UA and lead citrate staining were omitted.

            Figure 23.9 QDOT 525F(ab`)2 marker: (a) Without staining. (b) Negatively stained with uranyl acetate. Bar = 50 nm QDOT 525 marker after immunolabeling of E. coli sections: (c) Without enhancement. (d) For comparison, labeling with protein A-6 nm gold marker is shown. Bar = 100 nm   Figure 23.10 HQ SILVER enhancer applied to: (a) NANOGOLD marker (8 min). (b) Ultra-small gold marker (5 min). (c) R-GENT SE-EM enhancer applied to ultra-small gold marker (40 min). 


Bar = 100 nm

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 Figure 23.11 Danscher enhancer applied to: (a) NANOGOLD marker (35 min). (b) Ultra-small gold marker (30 min). (c) QDOT 525 marker (30 min). Bar = 100 nm  Figure 23.12 GoldEnhance-EM enhancer applied to NANOGOLD marker (30 sec). Bar = 100 nm  Caution: High background on immunolabeled ultrathin cryo-sections  Figure 23.13 Gold chloride treatment reduces gold size rather than leading to enhancement: (a) Untreated control. (b) Protein A-6 nm gold treated with 0.05% gold chloride for 30 min. Bar = 100 nm  Figure 23.14 The silver layer is sensitive to oxidation, e.g., during storage of labeled sections: (a) Image was taken directly after silver enhancement. (b) Image was taken several weeks after silver enhancement (same section, stored in humid ambient air). Bar = 100 nm  Figure 23.15 Gold chloride treatment (0.05%, 10 min, 20°C) stabilizes silver but leads to partial disintegration of the silver layer: (a) Untreated silver-enhanced control. (b) After gold chloride treatment, the silver layer decomposes into several smaller silver particles. Bar = 100 nm



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8. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

18.

Baschong W. and Stierhof, Y.-D. Preparation, use, and enlargement of ultrasmall gold particles in immunoelectron microscopy. Microsc. Res. Techn., 42, 66, 1998. Burry, R.W. Pre-embedding immunocytochemistry with silver-enhanced small gold particles, in Immunogold-Silver Staining, Hayat, M.A., ed., CRC Press, Boca Raton, FL, USA, 1995, 217. Danscher, G. Histochemical demonstration of heavy metals: A revised version of the sulphide silver method suitable for both light and electron microscopy. Histochem., 71, 1, 1981. Danscher, G. et al. Trends in autometallographic silver amplification of colloidal gold particles, in Immunogold-Silver Staining, Hayat M.A., ed., CRC Press, Boca Raton, FL, USA, 1995, 11. Dettmer, J. et al. Vacuolar H+-ATPase activity is required for endocytic and secretory trafficking in Arabidopsis. Plant Cell, 18, 715, 2006. Giepmans, B.N.G. et al. Correlated light and electron microscopic imaging of multiple endogenous proteins using quantum dots. Nat. Methods, 2, 743, 2005. Gilerovitch, H.G. et al. The use of electron microscopic immunocytochemistry with silver-enhanced 1.4 nm gold particles to localize GAD in the cerebellar nuclei. J. Histochem. Cytochem., 43, 337, 1995. Griffith, J.M. and Posthuma, G. A reliable and convenient method to store ultrathin thawed cryo-sections prior to immunolabelling. J. Microsc., 212, 81, 2003. Griffiths, G. Fine Structure Immunocytochemistry, Springer-Verlag, Berlin, Germany, 1993. Hainfeld, J.F. and Furuya, F.R. A 1.4-nm cluster covalently attached to antibodies improves immunolabeling. J. Histochem. Cytochem., 40, 177, 1992. Hainfeld, J.F., and Furuya, F.R. Silver-enhancement of Nanogold and undecagold, in Immunogold-Silver Staining, Hayat, M.A., ed., CRC Press, Boca Raton, FL, USA, 1995, 71. Harlow, E. and Lane, D. Using Antibodies, Cold Spring Harbor Laboratory Press, New York, 1999. Hayat, M.A. Principles and Techniques of Electron Microscopy, Cambridge University Press, Cambridge, UK, 2000. Hermann, R., Schwarz, H., and Müller, M. High precision immunoscanning electron microscopy using Fab fragments coupled to ultra-small colloidal gold. J. Struct. Biol., 107, 38, 1991. Holgate, C. et al. Immunogold-silverstaining: New method of immunostaining with enhanced sensitivity. J. Histochem. Cytochem., 31, 938, 1983. Lah, J.J., Hayes, D.M., and Burry, R.W. A neutral pH silver development method for the visualization of 1-nm gold particles in pre-embedding electron microscopic immunocytochemistry. J. Histochem. Cytochem., 38, 503, 1990. Leunissen, J.L.M. and Van De Plas, P. Ultrasmall gold probes and cryoultramicrotomy, in Immuno-Gold Electron Microscopy in Virus Diagnosis and Research, Hyatt A.D. and Eaton B.T., eds., CRC Press Inc., Boca Raton, FL, USA, 1993, 327. Pohl, K. and Stierhof, Y.-D. Action of gold chloride (“gold toning“) on silverenhanced 1 nm gold markers. Microsc. Res. Techn., 42, 59, 1998.


616 19.

20. 21. 22. 23. 24. 25. 26.


Sibon, O.C.M. et al. Ultrastructural localization of epidermal growth factor (EGF)receptor transcripts in the cell nucleus using pre-embedding in situ-hybridization in combination with ultra-small gold probes and silver enhancement. J. Histochem., 101, 223, 1994. Stierhof, Y.-D. et al. Yield of immunolabel compared to resin sections and thawed cryosections, in Colloidal Gold: Principles, Methods, and Applications. Hayat, M.A., ed., Academic Press Inc., San Diego, IL, USA, 1991, 87. Stierhof, Y.-D. et al. Direct visualization and silver enhancement of ultra-small antibody-bound gold particles on immunolabeled ultrathin resin sections. Scan. Microsc., 6, 1009, 1992. Stierhof, Y.-D. et al. Use of TEM, SEM, and STEM in imaging 1-nm colloidal gold particles, in Immunogold-Silver Staining. Hayat, M.A., ed., CRC Press, Boca Raton, FL, USA, 1995, 97. Stierhof, Y.-D., Humbel, B.M., and Schwarz, H., Suitability of different silver enhancement methods applied to 1 nm colloidal gold particles: An immunoelectron microscopic study. J. Electr. Microsc. Tech., 17, 336, 1991. Stierhof, Y.-D. and Schwarz, H. Labeling properties of sucrose-infiltrated cryosections. Scan. Microsc. Suppl., 3, 35, 1989. Tokuyasu, K.T. A study of positive staining of ultrathin frozen sections. J. Ultrastruct. Res., 63, 287, 1978. Weipoltshammer, K. et al. Signal enhancement at the electron microscopic level using Nanogold and gold-based autometallography. Histochem. Cell. Biol., 114, 489, 2000.

                       



3-D Electron Tomography of Cells and Organelles

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CONTENTS GENERAL INTRODUCTION .................................................................................... 621 1.

PRINCIPLES OF ELECTRON TOMOGRAPHY......................................... 622

2.

SUMMARY OF THE DIFFERENT STEPS ................................................... 624

3.

INSTRUMENTAL REQUIREMENTS ........................................................... 625 3.1. 3.2. 3.3. 3.4.

4.

PROTOCOLS .................................................................................................... 631 4.1. 4.2. 4.3. 4.4.

5.

Specimen Preparation ............................................................................... 631 Data Acquisition ....................................................................................... 633 Alignment, Reconstruction and Modeling................................................ 638 Short Protocol for Electron Tomography ................................................. 639

ADVANTAGES/DISADVANTAGES.............................................................. 641 5.1. 5.2.

6.

The Transmission Electron Microscope ................................................... 625 The Charge Coupled Device (CCD) Camera............................................ 627 High-Tilt Holders ..................................................................................... 628 Software.................................................................................................... 628 3.4.1. Data acquisition software............................................................ 629 3.4.2. 3-D data reconstruction software ................................................ 629

Advantages of 3-D Electron Tomography................................................ 641 Disadvantages of 3-D Electron Tomography ........................................... 642

WHY AND WHEN TO USE A SPECIFIC METHOD .................................. 644 6.1. 6.2.

Why Electron Tomography?..................................................................... 644 Why Electron Tomography on Resin Sections? ....................................... 644

7.

OBSERVED RESULTS .................................................................................... 646

8.

REFERENCES .................................................................................................. 648

 



621

GENERAL INTRODUCTION One of the techniques in electron microscopy that evolved rapidly in the past decade is transmission electron tomography. The method was first applied in 1970;1 however, only with increased computer power and once several of the technical challenges of the method were worked out,2 did electron tomography became a valuable and powerful tool. In the meantime, many electron microscopists realized that conventional electron micrographs merely were two-dimensional projections of three-dimensional structures and, in fact, an oversimplification of the actual structures in a cell. During the last few years, methods were developed to overcome some of the remaining limitations of electron tomography and focused on developing methods that provide a better understanding of the actual processes within cells, between tissues and in organs. The use of 3-D electron tomography in life sciences can be separated into several major fields of application, which have different requirements with respect to specimen preparation, the microscope and subsequent image processing. A possible distinction can be made between 1) cryo-electron tomography, 2) cellular tomography, 3) scanning transmission electron microscopy (STEM) tomography, and 4) energy filtering (EF) tomography. They all have their advantages and disadvantages. Cryo-electron tomography aims at imaging cryofixed and unstained material with lowdose transmission electron microscope (TEM)3 (see Chapter 12). Cryo-electron tomography is based on optimal specimen preservation, but is restricted to 3-D imaging of macromolecular complexes (>200 kDa), isolated cell organelles (< 500 nm), thin areas of cells (< 500 nm) or thin sections of cryo-fixed material.4,5 To date, there are no possibilities for specific labeling in cryo-electron tomography, but localization of large proteins with a characteristic and distinguishable shape can be mapped in their cellular context by template matching.6 Cellular tomography aims at reconstructing relatively large, unique structures by preferentially using high-pressure freezing (see Chapters 5, 6) and freeze-substitution (see Chapter 13) for specimen preparation.7-13 Often, several tomograms are combined to build 3-D reconstructions of even larger areas.14 Because of the dehydration and staining step, the specimen may be less well preserved than frozen-hydrated samples (see Chapters 1, 11). The method has, however, the advantage that large samples of cells and tissue can be processed easily. Furthermore, cellular components can be localized by specific labeling and the sections are more stable in the electron beam. Cellular tomography can also be applied to chemically fixed material and even to Tokuyasu cryo-sections for combination with immunogold labeling; however, one has to realize that structural artifacts are introduced.12 STEM tomography is a relatively new type of tomography.15-18 The technique has been shown to be very powerful for reconstructing highly diffracting specimens (e.g., crystalline material) that are not suitable for TEM imaging. When using a high-angle angular dark-field detector (HAADF), HAADF-STEM imaging collects images for which the contrast is more sensitive to differences in atomic weight. Therefore, for stained sections and for detecting ultra-small (gold) labels,19,20 STEM tomography has potential applications.18 To date, however, HAADF-STEM tomography is not often used in the life sciences.


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Electron tomography using energy filtering16 is an approach for imaging very thick sections (up to 1 µm) or cryo-sections (see Chapter 12). Often zero-loss filtering is used for cellular tomography. In addition, with sufficient electron dose, it may be possible to acquire 3-D images of areas containing a high concentration of a particular element, e.g., calcium.21 In this chapter, we will introduce the basic procedures and exemplify the methods that are used in electron microscopy facilities to obtain 3-D information on unique subcellular structures with nanometer-scale resolution and overview the continuing development of automated data acquisition programs as well as the improved user interface procedures.

1. PRINCIPLES OF ELECTRON TOMOGRAPHY  Electron tomography is a method to computationally generate a threedimensional (3-D) digital volume (the tomogram) from multiple 2-D projection images of the 3-D structure.  There are two assumptions that have to be taken into consideration when electron tomography is performed. 1 The first assumption is that the specimen does not change (shape, density) during the recording of the tilt series.

 For this reason, stained resin-embedded specimens have to be preirradiated, ensuring that the amount of specimen shrinkage during data collection is minimal.22

2 The second assumption is that the image is a true projection of the densities inside the specimen (projection requirement).

 This assumption does not hold for highly diffracting specimens (for those specimens STEM tomography is the method of choice).

 These assumptions set a limit to the  The use of energy filtering helps to meet thickness of the specimen that can be the projection requirement. observed. For very thick specimens (multiple), electron scattering events within the specimen can become too dominant.  For optimal results, the object must be  This is usually between 65o and +65o projected under an as wide as possible with an increment of 1o, resulting in a tilt range of viewing directions. series of 131 images.



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 The images need to be aligned and the  There are several procedures to compute reconstruction (called tomogram) can the 3-D reconstruction. Currently, the most widely used approach is resolutionbe computed. weighted back projection (WBP).7 In addition to this, there are other methods like the simultaneous iterative reconstruction technique (SIRT)23), the algebraic reconstruction technique (ART) or a dualaxis iterative algorithm from Tong et Midgley24  Once the tomogram is computed, complex structures in the interior of the specimen can be viewed in thin digital slices (often as thin as 3 to 10 nm), much thinner than can be accomplished with physical sectioning (50 to 80 nm). The obtainable resolution can be calculated by the following rule of thumb: three times section thickness divided by the number of images.

 This means that in a 350 nm-thick section with a recorded tilt series of 141 images (70 to +70o), a resolution of about 7.5 nm can be achieved.  3 × 350/141 = 7.5 nm

 Furthermore, in the 3-D digital  In this way, the structure of interest can volume, the object can be sampled then be analyzed and modeled. with image processing tools, e.g., segmented in any direction one wishes without being hampered by information from overlying structures in the region of interest.  With the current type of specimen  New approaches to achieve full, 180o holders, it is not possible to tilt the tilt, angles are under investigation.25 specimen to angles higher than 70o; at higher tilt angles the material holding the specimen blocks the field of view.  Due to the limitations of the angular tilt range, there is a lack of information at higher tilt angles. In the tomogram, this missing information is clearly visible in the shape of reconstructed high-density particles (e.g., gold beads).

 The beads will not be completely round, but will appear elongated in Z-directions. In the Fourier transform of the tomogram, the missing information can be seen as a missing wedge of information corresponding to the missing angular tilt range (see Section 5.2, Figure 24.18).

 The missing wedge can be reduced to a missing pyramid by rotating the grid over 90o, recording a second tilt series of the same area and combining the two tomograms into a dual-axis tilt reconstruction.26,27

 The improvement in resolution (especially in Z-direction) is very significant and results in a more isotropic resolution throughout the tomogram.


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2. SUMMARY OF THE DIFFERENT STEPS       A: The thick resin section with the structure of interest.

B: The projection images of the thick section. After recording the projection, images need to be aligned with respect to each other.

C: Individual slices from the computed tomogram.             


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D: Segmentation and annotation of the optical slices.

E: 3-D model of the structure.

Figure 24.1 (A-E) Schematic representation of the basic goal of performing electron tomography, namely the reconstruction of 3-D information from an object. (Sketch courtesy of Misjaël N. Lebbink.)

3. INSTRUMENTAL REQUIREMENTS 3.1. The Transmission Electron Microscope  To acquire 3-D information, electron tomography is performed on relatively thick specimens.   For imaging, sections of up to 500 nm thickness and a TEM with an acceleration voltage of at least 200 kV are necessary.


 Keep in mind that at a 60o tilt angle the apparent thickness of a 500 nm-thick specimen has doubled (1000 nm), and at tilt angles of 70o tripled (1500 nm).  Increase in specimen thickness results in increased scattering inside the specimen creating inelastic scattered electrons, which leads to blurring and reduced image contrast.

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 In many cases an imaging energy filter will be very helpful in generating highcontrast images. The images are taken at zero-loss, filtering out the inelastic scattered electrons that lost energy within the specimen.



 The goniometer should allow accurate  A goniometer that can tilt under cryo tilting of the specimen holder over a conditions is required for cryo-electron microscopy (see Chapter 12). large angular range.  For room temperature applications, dedicated tomography holders are available that allow tilting at high angles, sometimes in combination with dual-axis tilting.  Sophisticated acquisition packages (commercial and academic) are under constant development. They allow the automated tracking, focusing and recording of the area of interest.  The stability of the goniometer and the high-tilt holders allow holder calibration procedures28-30 to predict the movement of the holder at eucentric height in the microscope.

 Many of the automated tracking and focusing procedures are based on methods introduced in the early 1990s.31-33  Recording of a tilt series (131 images) can be performed within 45 minutes.

Figure 24.2 Tecnai-20 with LaB6 filament (FEI Company, Eindhoven, The Netherlands) at Utrecht University. This dedicated microscope is used solely for recording tilt series for 3-D electron tomography. The microscope is equipped with the automated data acquisition software Xplore 3-D (FEI Company, Eindhoven, The Netherlands). 


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3.2. The Charge Coupled Device (CCD) Camera  Before the advent of digital image recording, one of the most laborious and time consuming tasks of doing tomography was the processing and digitization of recorded tilt series from film. During the last decade, digital data acquisition using CCD cameras has become the most common type of data recording. The incoming electrons are converted to photons on a scintillator and then transmitted to the CCD chip either via fiber coupling or via a lens-coupled camera system.

 The size of the individual pixels on the CCD chip determines the final resolution (sampling distance) and the sensitivity of the camera.  The characteristics of the scintillator (type of material, thickness) in combination with the noise characteristic of the CCD chip determine the sensitivity of the camera system.  

 For cellular tomography on high contrast specimens it might be better to use a camera with smaller pixels and imperfect noise characteristics, whereas for cryo-electron tomography, with beam sensitive specimens and therefore low beam intensities, it is better to have a camera with larger pixels and very good noise characteristics. The quality of the digital cameras is improving continuously.

 Currently, CCD cameras for transmission electron microscopy will have between 10242 and 40962 pixels.  The increasing amount of pixels leads to large data sets close to 1 Gbyte in size.  Especially for iterative reconstruction algorithms this may lead to challenges with respect to computation and data storage.

Figure 24.3 The TemCam-F214 CCDcamera (TVIPS GmbH, Gauting, Germany) that records the projection images at the Tecnai-20 LaB6 microscope at the electron microscopy department of Utrecht University.


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3.3. High-Tilt Holders  The specimen grid holders must be able to tilt to high-tilt angles without obstructing the electron beam. The higher the achievable tilt angle the less loss in Fourier space and the smaller the so-called missing wedge artifact.  A second approach to reduce the size of the missing wedge is to rotate the grid by 90o and record a second tilt series of the same specimen area.26,27  To achieve these high-tilt angles, the material of the holder near the two sides of the grid is made as thin as possible to have a relatively large field of view, even at high-tilt angles (see Figure 24.4). Until recently, the in-plane rotation of the grid over 90o for recording the second tilt series had to be done manually. To facilitate the process of recording dual-axis tilt series dedicated specimen holders are now available, which allow in-plane rotation of the grid even when the holder is inserted into the electron microscope.

 Causing shadowing on the specimen.  These high-tilting angles are necessary to minimize the size of the missing wedge.  By combining the two tomograms, the missing wedge can be reduced to a missing pyramid. In our experience, it is more advantageous to record two tilt series from 60 to +60o and to combine them in one tomogram instead of recording one singletilt series from 70 till +70o.   Currently, we use the Fischione 2020 rotational tilt holder (Fischione Instruments, Pittsburgh, Philadelphia, USA) for this purpose.  They allow tilting angles up to +/80o.

Figure 24.4 The Gatan high-tilt holder (Gatan U.K., Abingdon, U.K.) with a clamping device for easy and firm clamping of the grid. 

3.4. Software  Not only hardware (see above) but  Three different types concerning the also software for automated data Three different steps: acquisition, data handling and data analysis is necessary. Several software packages are available for  Acquisition these purposes. Some are commercially available (meaning  Reconstruction expensive and without source code) and some are academic (meaning  Analysis and modeling much cheaper or even free and with  source code).



629

3.4.1. Data acquisition software  The main goal of the data acquisition software is to accurately collect a tilt series of digital images from a fixed location on the specimen. Irregularities in stage movement (x, y) and focusing (z) during the recording of the tilt series should be corrected. Many data acquisition software packages are available for image acquisition.

 Some of them are commercial packages (e.g., Xplore 3-D, FEI Company, Eindhoven, The Netherlands; EMmenu, TVIPS GmbH, Gauting, Germany; TEMography™, JEOL System Technology Co, LTD, Tokyo, Japan).  others have an academic origin and are freely available (e.g., TOM toolbox, MaxPlanck-Institute for Biochemistry, Martinsried, Germany; Serial EM, University of Colorado, Boulder, Colorado, USA; and Priism/IVE, UCSF, San Francisco, California, USA).

 The commercially available packages are stable and supported by a company but do not allow users to make adjustments in the software. The packages available through academia are more flexible and under constant development by the user community.

 Currently, in our department the Xplore 3-D package and the TOM toolbox are used for data acquisition at the electron microscopes.

3.4.2. 3-D data reconstruction software  The 3-D reconstruction software  In most cases, it takes less then an hour performs two tasks: alignment and to compute a dual-axis tilt tomogram. reconstruction. Currently, we use the IMOD package34 from University of Colorado (Boulder) to align the recorded projection images using fiducial markers. The tomograms are computed by means of the resolutionweighted back projection method.  For those occasions when the resolution-weighted back projection is less optimal (e.g., for very low contrast images), we use the ART or the SIRT modules available in the Xplore 3-D package. The resolutionweighted back projection algorithm implemented in IMOD is relatively fast.


 The advantage of a SIRT reconstruction algorithm is that the low frequency information is better resolved than with resolution-weighted back projection.

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 The ART and SIRT reconstruction algorithms have the disadvantage that they are computationally intensive and still lack the ability to generate dualaxis tilt tomograms.

 There is, however, a considerable reduction in ART and SIRT calculation times possible by using the computational power of graphical processing units or graphic cards on a standard desktop computer.

Figure 24.5 Two images of the user interface from the data reconstruction software package IMOD.34 This is one of the few packages that allow the reconstruction of double-tilt tomograms.


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4. PROTOCOLS 4.1. Specimen Preparation   





     Figure 24.6 Specimen preparation step scheme used for cellular tomography of cells and tissue.


632




 The quality of the tomogram produced with electron tomography completely depends on the quality of the sample in the microscope. The cellular structures under investigation must be prepared in a way that they represent a cellular state that is as close as possible to the native state. In order to achieve this, several cryofixation protocols were developed.  Specimen preparation protocols are under constant development and alternatives for traditional chemical fixation are used to overcome problems, such as displacement of the cellular proteins, conformational changes and denaturation of proteins. Also the shrinkage of tissues and cells during the fixation step12,35 and the loss of lipid content36 are important issues in developing new fixation protocols (see Matsko and Müller37 for a recent report).

 For specimens prepared by freezesubstitution, several protocols have been developed that include staining of cellular structures with heavy metals, like uranyl ions, lead ions or osmium tetroxide36 (see Chapter 13). For this type of specimen, the contrast mechanism is mainly scattering contrast.

 In our laboratory, the cryo-fixation  In our approach, section thickness method of high-pressure freezing normally ranges between 250 nm to (HPF) followed by freeze-substitution 500 nm. (FS) and resin embedding is often used (see Geerts et al8 for a detailed description). Briefly, the cryofixed cellular materials are embedded in a resin and thick sections are cut with an ultra-microtome using diamond knifes. The thick sections are collected on Formvar (or Pioloform)/carbon-coated copper grids.  In most of our studies on cellular arrangement, e.g., membrane continuities between organelles or connections between cellular compartments, 6, 10 or 15 nm gold particles are added randomly to both sides of the section. These gold beads function as fiducial markers during the alignment step that precedes the generation of the tomogram.


 In cases where specific immunogold labeling for the localization of particular proteins is performed, the specific gold label can sometimes be used also as a fiducial marker for the alignment step.

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 G = Golgi complex  ER = Endoplasmic reticulum  M = Mitochondria

Figure 24.7 Overview of a plasma cell in a 300 nm-thick section. The plasma cell was prepared by high-pressure freezing followed by freeze-substitution and embedding in Epon.   This procedure is especially suited to study membranes and their relationship.

Figure 24.8 Detail of the previous plasma cell showing an extensive endoplasmic reticulum (ER), a large Golgi complex (G) in which cisternae and the trans Golgi network (TGN), with numerous small vesicular structures, can be clearly recognized. 

4.2. Data Acquisition  The first prerequisite is a well aligned TEM with an accelerating voltage of at least 200 kV. The microscope alignment procedures are not described because they are outside the scope of this chapter.

 In our department we use a Tecnai 20 with a LaB6 filament equipped with a Tietz Temcam F214 CCD camera with 2048 × 2048 square pixels.

 Next, it is required that the CCD camera is properly installed and that the reference images (bias and gain reference images) are correctly recorded to produce high-quality digital images.

 For the installation and proper calibration of the CCD camera, we refer readers to the manuals provided by the manufacturer of the electron microscope and/or the camera.


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 When one acquires a manual tilt series, it becomes clear that the repetitive actions needed to keep the area of interest centered and focused are time consuming. Area selection, focusing and tracking has to be done by intense interaction between the user and the computer program controlling the microscope. Once digital imaging became a commodity on most electron microscopes, automation was developed that replaced the human operations to center and focus.  In 2001, it was noted that imperfections of the tilting movement of the goniometer (see Figure 24.9) were very reproducible.28

Figure 24.9 The holder calibration curves for the Gatan high-tilt holder that can be used for automatic recording of data sets. For visualization, purposes the thickness of the calibration line is adjusted in Adobe ®  Photoshop .

 Calibration curves were used to  Today automated tomography data predict image and focus shifts and to acquisition software packages are available compensate for them automatically on a variety of TEMs. during data acquisition. Variations and improvements on this basic idea were realized and further improved in several packages (UCSF package 2004,29 TOM toolbox, Martinsried, 2005, SerialEM.30) 











 An important concept in automated data acquisition is the “optimized position” concept. From a theoretical point of view, any point on the specimen undergoes a circular motion, with the center of the circle lying on the tilt axis (a line in horizontal direction determined by the goniometer) and the radius of the circle being the distance between the point and the tilt axis.

635  Theoretically, the radius of the circular movement is then minimal.  By setting the specimen at eucentric height, the position of the specimen within the goniometer is changed until the height of the specimen and the tilt axis are in the same plane.

 However, in practice there still will be  The distance between these two axes, the movement visible when the misalignment, can be several m. goniometer is tilted. This is due to the fact that the position of the tilt axis (horizontal) does not intercept with the optical axis of the microscope (vertical) that defines the area that is imaged on the center of the CCD image.  The major effects of this misalignment are a focus change and translational shift when the specimen is tilted.

 However, focus changes and translational shifts can be corrected by adapting the objective lens and the image shift controls of the microscope.

 Fortunately, automated correction of the misalignment is carried out by adjusting the optical axis of the microscope (vertical) to coincide with the tilt axis of the goniometer (horizontal). To carry this alignment through, a number of basic calibrations need to be performed.

 The purpose of these calibrations is to give a physical meaning to the pixel size of the CCD camera (the magnification) and to calibrate the image shift, stage shift and focus shift controls

 Apart from these general calibrations there are two tomography-specific calibrations, those of the tilt axis (measuring the optimized-position shift — the distance between the optical axis and the tilt axis) and measuring the remaining focus changes and translational shifts of the specimen when it is tilted.

 Used to predict the image shift and focus changes that are needed during tilt-series acquisition to keep the area of interest in the center of the image.  These calibrations have to be checked once a month.


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 When these prerequisites are fulfilled, recording a tilt series is a straight forward procedure that can be performed within an hour.  As stated before, in our laboratory we image specimens prepared with cryofixation and freeze-substitution. We use stained resin-embedded sections of 250 to 500 nm thickness, often decorated with fiducial markers (gold beads in the size range of 5 to 15 nm) on both sides of the specimen.

 For optimal alignment, we need 10 to 15 gold particles per image.  The required dilution of the gold particle solution can be checked by applying a droplet of a particular dilution on bare grids and examining them in the microscope at the specific magnification that will be used for recording the tilt series

 The specimen grid is tightly clamped  This is absolutely necessary into a high-tilt holder (single-tilt or recording proper tilt series. rotation holder) and carefully inserted into the goniometer of the microscope.

for

 Next, the area of interest has to be set  Eucentricity has to be checked every at eucentric height. time before recording a new tilt series, either by hand or with the data acquisition software.  Before recording the tilt series, it also has to be checked (by tilting the holder) on how far the area of interest can be tilted without any obstructions from the grid bars or the holder itself.  Next, the intensity of the beam on the  This is necessary to prevent overspecimen is checked at the zero illumination of the area of interest during degree tilt angle. the recording of the tilt series.  Keep in mind that the light intensity on the CCD screen depends on the tilt angle of the sample; at high-tilt angles the section thickness increases considerably resulting in much weaker image intensity on the CCD chip.  Finally, the actual recording of the projection images can start. In general, it takes about 45 minutes to record a complete series of projection images.



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Figure 24.10 User interface of the FEI automated data acquisition software where data acquisition parameters can be adjusted.

Figure 24.11 User interface showing the tracking and focusing images and the corresponding cross-correlation images that are used for automated focusing and tracking of the area of interest.

Figure 24.12 The user interface that allows visualization of the recorded tilt series. The scale bar, the tilt angle and the tilt axes are viewed and stored as tiff images.

Figure 24.13 The goniometer of the Tecnai-20 (FEI Company).


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4.3. Alignment, Reconstruction and Modeling  The series of projection images (tilt  For the reconstruction process, several series) are stored for subsequent academic and commercial packages are computation of the tomogram. available.  The resolution-weighted back project-  WBP is a fast reconstruction procedure. tion algorithm,23 which is also implemented in the IMOD package is a widely used method for computing the tomogram. For a more detailed description of this package, we refer to Kremer et al.34  In short, the individual projection images of the recorded tilt series show small image shifts from one projection image to the next. The first alignment step in IMOD reduces the image shifts from one image to the next by means of a cross correlationbased alignment of the individual images.  In the second alignment step, fiducial markers present in the projection images (of the gold particles) are selected and used for a more accurate alignment of the projection images. Then the tomogram can be computed using the resolution-weighted back projection method. Also available in IMOD is the possibility to generate dual-axis tilt tomograms. After the tomogram is calculated, the analysis of the recorded data set can start.

 For a more elaborate description we refer to the excellent manual and the Web site of the University of Colorado (http://bio3d.colorado.edu/imod/).

 In the IMOD package, several options are available for modeling. There is a contour drawing mode (most often used), a thresholding tool for automatic selection and drawing of contours and contour volume rendering options.

 So far, all the steps in the acquisition process can be performed without specific knowledge of the object under study.  However, for interpretation of the tomogram and modeling, specific knowledge of the object is absolutely necessary.  In many studies, using the contour mode, the extent to which membranous compartments are connected to other membranous compartments is investigated, both in the exocytic as well as in the endocytic pathways.38,39


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 An illustrative example is the connection between the ER and the newly originating peroxisomes.40  The contour drawing mode is, however, prone to the interpretation by the investigator. Therefore, efforts are now being undertaken to find computational methods for automated pattern recognition by means of thresholding and template matching by computers.18,41

Figure 24.14 Projection image of a 300 nm-thick plastic-embedded section with randomly applied fiducial gold markers (10 nm), which can be seen as dense black dots all over the section.

4.4. Short Protocol for Electron Tomography 1. Before starting data acquisition check  Also the TEM basic calibrations and the the general microscope conditions: TEM tomography calibrations must have Beam tilt pivot points, tomo rotation been carried out. center, beam tilt calibrations, image (beam) calibration, image shift pivot points, image shift for the magnification range. 2. Check the CCD dark image and gain  For the dark image correction, close the correction. The fully corrected image screen and record the dark image. should appear homogenously grey.  For the gain correction, remove the specimen and illuminate the CCD evenly with a number of counts not too different from the working conditions later. 3. Load the sample and locate the area of interest. 4. Adjust eucentric height manually.


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5. Focus. 6. Center the condenser aperture. 7. Adjust the gun conditions. 8. Align rotation center. 9. Correct objective astigmatism and focus image.

Figure 24.15 User interface of Xplore 3-D for checking light intensity and astigmatism. 10. Check for the maximum tilt angles without blocking the area of interest. 11. Adjust the CCD settings for search, focus, exposure and tracking. 12. Set the parameters for recording a tilt series.

Figure 24.16 User interface of Xplore 3-D for setting the data acquisition parameters for automated recording and storage of the tilt series.

13. Provide a unique name for storage of the image series. 14. Check if the focusing and tracking steps are performed well. 15. Check the recorded tilt series in visualization mode.



16. Align the individual images with IMOD.

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projection

17. Calculate the tomogram with the weighted back projection method in IMOD. 18. Analyze the tomogram. 19. Perform the contouring and/or surface  Amira is available from Mercury rendering in IMOD or Amira. Computer Systems SAS, 33708 Merignac Cedex, France.

5. ADVANTAGES/DISADVANTAGES 5.1. Advantages of 3-D Electron Tomography  The produced dataset contains  To achieve the best possible Z-resolution projection images of the same in transmission electron microscopy position under different angles. studies, sections need to be as thin as possible. In the recorded projection image, however, structures obscure each other and the achieved Z-resolution is never better than the section thickness.  The third dimension in the section is  By the use of the back projection restored. method. In this way a “virtual” 3-D block is created and individual images can be retrieved from this block and used for analysis.  The Z-resolution obtained is  In general, the Z-resolution of a determined by the number of recorded tomogram is approximately 10 times better projection images and the section then in a single projection image. thickness.  The 3-D digital volume: The object can be sampled with image processing tools, e.g., segmented in any direction one wishes without being hampered by information from overlying structures in the region of interest.


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Figure 24.17 A. The information in a projection image of a 300 nm-thick plastic section displays low Z-resolution and is obscured by overlying structures and, therefore, difficult to interpret.  Note: The increased resolution in all directions and the amount of detail present in the slice. B. Single optical slice extracted from the 3-D reconstruction of the thick section (300 nm) shown above. N = Nucleus ER = Endoplasmic reticulum with ribosomes on the membranes G = Golgi S = Spindle

5.2. Disadvantages of 3-D Electron Tomography  The current types of specimen holders  Due to this limitation of the angular tilt prevent tilting the specimen to angles range, there is a lack of information at the higher than +/70o. higher tilt angles.  In the Fourier transform of the tomogram, the missing information can be seen as a missing wedge of information corresponding to missing angular tilt range.



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Figure 24.18 Schematic drawing of the missing wedge information in Fourier space. The gray area contains information that is not present in the reconstruction due to the limited tilt angles within an electron microscope.

 Have to combine two perpendicular  The improvement in Z-direction is tomograms into a dual-axis tilt significant and results in a more isotropic reconstruction.26,27 resolution throughout the tomogram.  No real 3-D labeling is possible.

 Many EM studies are performed on resin-embedded samples. This usually provides good morphological quality, but prevents specific localization studies because the penetration of antibodies into the section is not possible. So immunolabeling can be performed only at the surface of the resin-embedded section.

Figure 24.19 Comparison of the same area (X/Z) in a single and double-tilt tomogram. In the single-tilt tomogram (top), a clear distortion/elongation in the Z direction is visible. The double-tilt tomogram (bottom) shows improved Z-resolution.


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6. WHY AND WHEN TO USE A SPECIFIC METHOD 6.1. Why Electron Tomography?  With transmission electron microscopy of thin sections (40 to 70 nm), unique subcellular structures are studied and high X, Y resolution can be obtained. However, many fine details (especially in Z-direction) are obscured by overand underlying structures in the section.  3-D transmission electron tomography can provide unmistakable answers to these questions with a resolution especially in Z that is 10 times better then obtainable with the classical methods.  By performing 3-D tomography, it was possible to gain new insight and generate new hypotheses for many biological processes, such as the generation of the peroxisome,9 COP II localization,42 connections between cisternae in the Golgi39,43 transport in the endo- and exocytic pathways, nuclear organization and many more.

 This makes it difficult to give unambiguous answers to certain questions, like, for instance, is there a connection between organelles (direct open contact or MCS (membrane contact sides) or between subcompartments of an organelle (direct connections between cisternae of a Golgi system)?

 Resolution with light microscopy is at best, 200 nm.

6.2. Why Electron Tomography on Resin Sections?  Performing tomography on resin sections has a number of advantages.  First, the specimen preparation steps  Many EM labs can perform the basic are relatively simple. steps of fixing (either chemical or the more advanced high-pressure freezing followed by freeze-substitution), resin embedding, and sectioning and contrasting of sections.  Second, the presence of abundant amounts of heavy metals (uranium, osmium, lead) in the section results in a high signal-to-noise ratio (good contrast), making it easy to recognize structures and focus on the specimen.



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 Third, it is even possible to determine  When enough specific gold labels are protein localization on metacrylate- present on the section surface, they can be used for alignment of the projection images embedded material. and tomograms were specific labeling on top of the section can be studied and analyzed.  Fourth, the presence of resin provides rigidity to the specimen. After the initial shrinkage of the resin under the electron beam, the section is rather stable allowing the recording of a large number of projection images with small incremental values. This results in a resolution that is impossible to obtain with cryotomographic methods. The total electron dose that can be used on the specimen is much higher than in cryotomography.

 Initial volume shrinkage can be up to 30%.

 Fifth, it is easy to reduce the so-called missing wedge artifact into a missing pyramid by rotating the grid by 90o and recording a second tilt series of the same area of interest. The stability of the specimen and the unique nature of the object allow the microscopist to find the region of interest after rotating the grid.

 The two computed tomograms can be combined into one double-tilt tomogram. The resolution (especially in Z-direction) benefits considerably from this. Rotating can be done in the microscope (rotation high-tilt holder) or outside the microscope.


 In cryo-EM, the total dose must be divided over all the recorded images. This results in fewer images with a much lower signal-to-noise ratio (see Chapter 12).

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7. OBSERVED RESULTS  Figure 24.20

 The whole process of doing 3-D electron tomography is exemplified on a cellular organelle (a multivesicular lysosome).  A-C: Representative images of the first stack of projection images recorded from 65 to +65o with an increment of 1o Shown are the projection images at 60o (A), 0o (B) and +60o (C).  D-E: Images of the stack perpendicular to the first one from the same area at 60o (D), 0o (E) and +60o (F).  G: The combined tomogram. Because it is constructed from two independently constructed tomograms, there is a region that contains singletilt information (upper right part) and one that contains double-tilt information (lower left part).  H-I: Two representative optical slices from the double-tilt tomogram illustrating the increased resolution of the tomogram compared to the zero tilt images in B and E.

 Figure 24.21 (see colour insert following page )


 A: Optical slice with some contours of the different suborganellar structures in the multivesicular lysosome. The contouring was done with the IMOD software package.  B: A surface rendered model that was made from the contouring of the multivesicular lysosome. For demonstration, only a few of the subcellular structures are visualized.



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8. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

Crowther, R.A., DeRosier, D.J., and Klug, A. The reconstruction of a threedimensional structure from its projections and its applications to electron microscopy, P. Roy. Soc. Lond. A Mat., 317, 319, 1970. Koster, A.J. et al. Perspectives of molecular and cellular electron tomography, J. Struct. Biol., 120, 276, 1997. Medalia, O. et al. Macromolecular architecture in eukaryotic cells visualized by cryoelectron tomography, Science, 298, 1209, 2002. Al-Amoudi, A., Norlen, L.P.O., and Dubochet, J. Cryo-electron microscopy of vitreous sections of native biological cells and tissues, J. Struct. Biol., 148, 131, 2004. Leis, A.P. et al. Cryo-electron tomography of biological specimens, IEEE Signal Proc. Mag., 23, 95, 2006. Frangakis, A.S. and Förster, F. Computational exploration of structural information from cryo-electron tomograms, Curr. Opin. Stuct. Biol., 14, 325, 2004. Frank, J. Electron Tomography: Three-Dimensional Imaging with the Transmission Electron Microscope. Plenum Press, New York, London, 1992. Geerts, W.J.C. et al. Electron microscopy tomography and localization of proteins and macromolecular complexes in cells, in Protein-Protein Interactions. A Molecular Cloning Manual, Golemis, E.A. and Adams, P.D., eds,. Cold Spring Harbor Laboratories Press, New York, USA, 2006, 715. Geuze, H.J. et al. Involvement of the endoplasmic reticulum in peroxisome formation, Mol. Biol. Cell, 14, 2900, 2003. Horowitz, R.A. et al. The three-dimensional architecture of chromatin in situ: Electron tomography reveals fibers composed of a continuously variable zig-zag nucleosomal ribbon, J. Cell Biol., 125, 1, 1994. Marsh, B.J. et al. Direct continuities between cisternae at different levels of the Golgi complex in glucose-stimulated mouse islet beta cells, Proc. Nat. Acad. Sci. USA, 101, 5565, 2004. Murk, J.L.A.N. et al. Influence of aldehyde fixation on the morphology of endosomes and lysosomes: Quatitative analysis and electron tomography, J. Microsc., 212, 81, 2003. Tomova, C. et al. New comprehension of the apicoplast of Sarcocystis by transmission electron tomography, Biol. Cell, 98, 535, 2006. Marsh, B.J. Reconstructing mammalian membrane architecture by large area cellular tomography, Methods Cell Biol., 79, 193, 2007. Midgley, P.A. et al. Probing the spatial distribution and morphology of supported nanoparticles using rutherford-scattered electron imaging, Angew. Chem. Int. Ed. Engl., 41, 3804, 2002. Midgley, P. and Weyland, M. 3D electron microscopy in the physical sciences: The development of Z-contrast and EFTEM tomography, Ultramicroscopy, 96, 413, 2003. Ziese, U. et al. Three-dimensional localization of ultrasmall immuno-gold labels by HAADF-STEM tomography, J. Struct. Biol., 138, 58, 2002. Yakushevska, A. et al. Comparison of TEM and STEM tomography in cell biology, J. Struct. Biol , 159, 381, 2007. Otten, M.T. et al. High-angle annular dark-field STEM imaging of immunogold labels, Scanning, 14, 282, 1992. Stierhof, Y.-D. et al. Use of TEM, SEM, and STEM in imaging 1-nm colloidal gold particles, in Immunogold-Silver Staining: Principles, Methods, and Applications, Hayat, M.A., ed., CRC Press, Boca Raton, FL, USA, 1995, 97. Leapman, R.D. Detecting single atoms of calcium and iron in biological structures by electron energy-loss spectrum-imaging, J. Microsc., 210, 5, 2003.



22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43.

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Luther, P.K. Sample shrinkage and radiation damage, in Electron Tomography. Three-Dimensional Imaging with the Transmission Electron Microscope, Frank, J., ed., Plenum Press, New York, London, 1992, 39. Gilbert, P. Iterative methods for the three-dimensional reconstruction of an object from projections, J. Theor. Biol., 36, 105, 1972. Tong, J. and Midgley, P. A novel dual-axis reconstruction algorithm for electron tomography, J. Physics: Conf. Ser., 26, 33, 2006. Kamino, T. et al. Application of a FIB–STEM system for 3D observation of a resin-embedded yeast cell, J. Electr. Microsc., 53, 563, 2004. Penczek, P. et al. Double-tilt electron tomography, Ultramicroscopy, 60, 393, 1995. Mastronarde, D.N. Dual-axis tomography: An approach with alignment methods that preserve resolution, J. Struct. Biol., 120, 343, 1997. Ziese, U. et al. Automated high-throughput electron tomography by pre-calibration of image shifts, J. Microsc., 205, 187, 2002. Zheng, Q.S. et al. An improved strategy for automated electron microscopic tomography, J. Struct. Biol., 147, 91, 2004. Mastronarde, D.N. Automated electron microscope tomography using robust prediction of specimen movements, J. Struct. Biol., 152, 36, 2005. Dierksen, K. et al. Three-dimensional structure of lipid vesicles embedded in vitreous ice and investigated by automated electron tomography, Biophys. J., 68, 1416, 1995. Koster, A.J. et al. Automated microscopy for electron tomography, Ultramicroscopy, 46, 207, 1992. Fung, J.C. et al. Toward fully automated high-resolution electron tomography, J. Struct. Biol., 116, 181, 1996. Kremer, J.R., Mastronarde, D.N., and McIntosh, J.R. Computer visualization of three-dimensional image data using IMOD, J. Struct. Biol., 116, 71, 1996. Szczesny, P.J., Walther, P., and Müller, M. Light damage in rod outer segments: The effects of fixation on ultrastructural alterations, Curr. Eye Res., 15, 807, 1996. Humbel, B.M. and Schwarz, H. Freeze-substitution for immunochemistry, in Immuno-Gold Labeling in Cell Biology, Verkleij, A.J. and Leunissen, J.L.M., eds., CRC Press, Boca Raton, FL, USA, 1989, 115. Matsko, N. and Müller, M. Epoxy resin as fixative during freeze-substitution, J. Struct. Biol., 152, 92, 2005. Murk, J.L.A.N. et al. Endosomal compartmentalization in three dimensions: Implications for membrane fusion, Proc. Nat. Acad. Sci. USA, 100, 13332, 2003. Trucco, A. et al. Secretory traffic triggers the formation of tubular continuities across Golgi sub-compartments, Nat. Cell Biol., 6, 1071, 2004. Murk, J.L.A.N. 3-D analysis of endosomes, lysosomes and peroxisomes. PhD thesis, Utrecht University, Utrecht, The Netherlands, 2004. Lebbink, M.N. et al. Template matching as a tool for annotation of tomograms of stained biological structures, J. Struct. Biol., 158, 327, 2007. Zeuschner, D. et al. Immuno-electron tomography of ER exit sites reveals the existence of free COPII-coated transport carriers, Nat. Cell Biol., 8, 377, 2006. Polishchuk, R.S. and Mironov, A.A. Structural aspects of Golgi function, Cell. Mol. Life Sci., 61, 146, 2004.



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FINAL CONSIDERATIONS The publication of this handbook is a wonderful satisfaction for a scientist engaged throughout his long life in the development of instrumentation and methodologies for research in biological ultrastructure. It is proof that some dreams may become reality and that “cryo” is still in a dynamic and fruitful state. twenty years ago, Steinbrecht and Zierold10 edited the first compilation in this field. Their book already demonstrated the incredible advances since the early 1950s. At that time, high resolution transmission electron microscopes were available for research.3,7 The first chapters of this book document the current state of the art. It’s a story of success. A dream became reality; the dream to look directly by microscopic observation inside the basic structures of life. The way to do this starts with the proper vitrification of macromolecules, cells in suspension or tissues. Both instrumentation and methodology for freezing, post- and prefreezing steps of such preparations have now reached a state of perfection, which makes it almost an easy task and at the very least a feasible one. Progress in cryo-technology had an important side effect for science — the tiny water molecule was brought again into the forefront. It was astonishing that most scientists — even biologists — for a long period of time had no real interest in this simple compound, albeit with a complex behaviour, made of two hydrogen and one oxygen atom. Really astonishing, because it was obvious and well known that water molecules represent a major component of our huge universe. Astonishing also because more than half of our earth’s surface is covered by oceans. Astonishing with respect to the community of biologists because any student knows that cells, plants and animals, including man, contain more water than other components. Nevertheless, water was ignored successfully. It was hampering structural studies in the light microscope and had to be removed completely to allow wax (paraffin) embedding. A good penetration of the dehydrating agent was only possible after poisoning the cells to make their membranes leaky and penetration possible. Those were useful methods, but also a solid basis for plenty of artefacts. Concerning ultrastructural research, it was obvious that proper “vitrification” (see Chapter 1) within milliseconds was the only way to stabilise all components of the sensitive, mixed plasmatic phases, including also small molecules and rapidly moving ions. But there was a general scepticism by the experts in physical chemistry as to whether real vitrification of pure water and diluted aqueous solutions was actually possible. In 1980/81, two independent teams led by Brüggeller and Dubochet demonstrated this possibility.1,2 This was a breakthrough of great importance that opened the door to answering a lot of questions. If water has no time to aggregate into crystals, then all other components must also be held in their original position. Proper vitrification, therefore, justifies the considerable effort carried out to develop cryoultramicrotomy and perform diffraction analysis of ultrathin cryo-sections in the “frozen-hydrated state.” That now really makes sense.9


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Most progress discussed above was achieved against all odds. Similar obstacles were encountered in the development of the electron microscope. Gabor, as a member of the team around Knoll at the Techniche Hochschule Berlin, wrote the most impressive and competent report about the early days of this development.3 I cite in the original language: “Niemand konnte anhand der damals verfügbaren Unterlagen vorausahnen, dass das Elektronenmikroskop so erfolgreich sein würde, wie es sich später erwies”. He continued, “Wir wollen im Voraus die Moral aus dieser Geschichte ziehen: Selbst in der Wissenschaft ist es oft besser, Mut zu besitzen, als gescheit zu sein”. Translated into English: “At that time, nobody could according to the available data predict that the electron microscope would be such a success as it turned out later on” and “We draw in advance the conclusion from this story: even in science, it is often better to be courageous than to be very intelligent.” I noticed with much pleasure the cynical comments of all the honourable old professors in scientific journals at the time. They widely discussed this useless crazy new instrument. These comments would be worth a historical study — as an example for the old slogan, “errare humanum est.” I would like to recall a few other examples of such errors: a. Ultramicrotomy: No one believed it would be possible to reproducibly cut ultrathin sections in the nanometer-range suited for the EM. Neither precision mechanics nor knife materials nor fixatives nor embedding media seemed to be achievable. Nevertheless, among others, Porter and Blum6 did it. b. High-pressure freezing: Moor and Riehle5 were successful. I cite. Moor4: “The interest of the audience was not overwhelming because everybody thought that this approach is over sophisticated and unnecessary” – what an error! High pressure freezing provided the first pictures of well-frozen ultrastructures and a world of new information. c. Finally, my own experience in cryo-ultramicrotomy:9 Nobody wanted to believe that sectioning at 180°C would be successful. A group of smart, experienced cryotechnologists from industry visited our prototype FC4 and stated that such a set-up with direct LN2 cooling and an open top could never work (it was already running and not so badly). One of these fellows stated that we probably had manipulated the temperature indicators. He wet his fingertip and felt if the knife holder was really cold (it was approximately 150°C). I was silent and smiling when he yelled because he suffered a severe burn. I noticed something else, all these “crazy developments, which never would work” were achieved by rather young scientists, often students. The first EM was built by a “young crew” around the also youthful assistant. Knoll. His team was made up only of students of the TH Berlin. Jacques Dubochet2 was young as well as his team at the EMBO, Heidelberg. Nevertheless, they achieved and documented proper vitrification of pure water. Porter and Blum6 were young fellows when they built (at the Rockefeller Institute in New York), the first ultramicrotome, which was clearly suited for routine work. That was the Volkswagen in ultramicrotomy and served generations of scientists over decades, an astonishingly simple apparatus. Moor and Riehle5 as young scientists also did an excellent job at the ETH Zürich. I started as a young student in the third semester on an EM and built my first ultramicrotome.8 I wanted, together with my brother Peter (also a student), to have a look into cells. In general, youngsters have the courage necessary to enter into risky projects and have the drive needed for success. The slogan:“Doing


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the Impossible” fits to their kind. One should be careful with this precocious resource and try to keep these youngsters instead of firing them after some years of successful engagement. The United States tenure track system seems a good solution, which we lack in Europe. I want to make some further comments on “cryo” analyses (and on expensive research in general): “Enthusiasm cannot substitute for Money.” This means that efficient cryowork in many —if not most — sectors needs some money. Electron microscopes (equipped with a cryo-stage, low dose, possibly filter lenses), cryo-ultramicrotomes (with cryo-diamonds, antistatic devices, etc.), a high-pressure freezer, cryotransfer units, automated freeze-substitution and freeze-drying units are not available for nothing. The same applies to running costs. This is a threat for universities and grant-giving organisations. Money is rare — especially in Europe and the Southern Countries. This is a severe problem for cryo. Our colleagues in the US and Canada are in a splendid situation. They also complain that the budget decreases every year. But a true comparison reduces my compassion immediately to nothing. Certainly there are some low-cost set-ups like home-built plunging systems working with a rubber belt like David’s catapult to kill Goliath. But such instruments are rather the exception. There are some examples in which lack of money may provoke inventions that end in a Nobel Prize award. The one most often cited is about the Austrian analytical chemist Fritz Pregl who earned the Nobel Prize for the foundation of quantitative organic microanalysis in 1923. It was said that he was forced to this discipline simply by lack of money. I do not believe this. The same rumour concerns the discipline of theoretical physics in Göttingen (Germany). Maybe, but I want to warn governments from reducing budgets of universities and research organisations to increase the number of Nobel Prizes that such an inverse relationship will certainly not work. It would also be dangerous to split the old fashioned universitas litterarum into expensive and inexpensive disciplines. Then cryo will come to an end together with other expensive disciplines. Markl, former president of the big German research organisation (Deutsche Forschungsgemeinschaft) stated in an official meeting about the financial situation, that we must avoid supporting, for financial reasons, more low-cost projects. Then we would provoke the degeneration of research into “Mickey Mouse research.” I guess personally that plenty of Mickey Mouses are already at work in our scientific community. These are scientists who calculate exactly the probability of success and the time needed. Only 100% success rates and short periods of time are wanted. They should leave the university and change over to a commercial employment. In my opinion, they will not do this and also will not be wanted in this alternative area — sorry. But, I again warn governments. There is a pitfall, a kind of sin. The “splitting of money into fractions of “useful” (applied) and “useless” (basic) research” is a horrible mistake. Useful research is most often light-weight research that serves commerce and governments. Useless research is heavy-weight research, which has no immediate visible and predictable influence on commerce and, therefore, income taxes for the government. The claim is that this useful research increases the welfare of all citizens. Most citizens feel that this is a striking argument. The truth is just the contrary.


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The economic success in the United States resulted to a great extent from the generous support of the so-called “useless” basic research. Only such basic research opens completely unexpected new areas for commerce, industry and other avenues in a country. Internationally, well-known scientists made very clear statements and warned the Austrian Government. I cite two colleagues working in Vienna in prominent positions. (1) Freissmuth, pharmacologist and director of a big Institute of the Medical University in Vienna, stated, “You cannot save (basic) research.” (2) Penninger, pathologist and director of the Institute of Molecular Biotechnology (Austrian Academy of Sciences) just recently stated in an official discussion of the broadcast ORF1, “If the government saves money on basic research it will change our university into a museum.” This is perfectly valid for cryo-work. With an old EM, a historical ultramicrotome and an old evaporation unit, you may impress plenty of visitors. But you are unable to carry out state-of-the-art cryo-work. You degenerate into a “Mickey Mouse,” according to Markl. Since I wish all young colleagues a satisfying and fruitful career in this fascinating field, I have made some comments concerning a sufficient amount of money for this type of research. I repeat: “Enthusiasm in most situations cannot substitute for money.” A strong financial basis is of key importance for success besides courage, intelligence and the always needed drive. In this sense, I wish all fellow workers, especially the young ones, “Good luck in the fascinating cryo-work.”

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Brüggeler, P. and Mayer, E. Complete vitrification of pure liquid water and diluted aqueous solutions, Nature (London), 288, 569, 1980. Dubochet, J. and McDowall, A.W. Vitrification of pure water for electron microscopy, J. Microsc. (Oxford), 124, RP3, 1981. Gabor, D. Die Entwicklungsgeschichte des Elektronenmikroskopes, Elektrotechn. Zschr., 78, 522, 1957. Moor, H. Theory and practice of high pressure freezing, in Cryotechniques in Biological Electron Microscopy, Steinbrecht, R.A. and Zierold, K., eds., Springer Verlag, Berlin, Heidelberg, Germany, 1987, 175. Moor, H. and Riehle, U. Snap-freezing under high pressure: A new fixation technique for freeze-etching, in Proc. 4th Europ. Reg. Conf. Electron Microsc., Bocciarelli, S., ed., 1968, 33. Porter, K.R. and Blum, J. A study in microtomy for electron microscopy, Anat. Rec., 117, 685, 1953. Ruska, E. Die frühe Entwicklung der Elektronenlinsen und der Elektronenmikroskopie, Acta historica Leopoldina Nr. 12. Deutsche Akademie der Naturforscher, Halle/Salle, 1979. Sitte, H. Ein einfaches Ultramikrotom für hochauflösende elektronenmikroskopische Untersuchungen, Mikroskopie (Wien), 10, 365, 1955. Sitte, H. Advanced instrumentation and methodology related to cryoultramicrotomy: A review, Scan. Microsc. Suppl., 10, 387, 1996. Steinbrecht, R.A. and Zierold, K. Cryotechniques in Biological Electron Microscopy. Springer Verlag, Berlin, Heidelberg, Germany, 1987.



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A  Algebraic reconstruction technique  A reconstruction algorithm that (ART) compares the differences between the reprojections of a tomogram and the measured data, one image at a time, and corrects the volume iteratively until a given stopping criterion is fulfilled.  Amorphous ice  Solidified water devoid of crystals, also called vitreous ice.  Analytical probes  Electron beam or ion beam that interacts with specimens are analytical probes.  Antibody  Immunoglobulins that recognize specific antigens.  Antifreezing agent  A substance that is added to water to lower its melting point.

B  Bar  Basic calibrations  Blocking buffer

 Blotting  Boltzmann constant, k

 Measure of pressure: 1 Bar = 105 Pa = 752.5 Torr = 0.99 atm.  Measured curves describing the movement of a high tilt holder in x, y and z during a tilt range.  Used to prevent undesired binding of antibodies to biological structures and resin (usually interactions of electrostatic and hydrophobic nature).  Removal of excess fluid from the grid prior to vitrification in liquid ethane.  The physical constant relating temperature to energy; experimentally determined as 1.38 × 10 to 23 joules/Kelvin.

C  Celsius

 Anders Celsius 1701–1744, Swedish astronomer, devised the Celsius (oC) temperature scale (Dictionary of Science and Technology, Academic Press, San Diego, CA, 1992).

 CEMOVIS

 Technique allowing observation of sections of fully frozen-hydrated samples under an electron beam (in a cryo-electron microscope).


660  Charge-coupled device (CCD)


 An image sensor comprising an integrated circuit and a linked array of radiation-sensitive capacitors.  Chatter  One of the major cutting artifacts. It is a periodic variation of section thickness at the µm scale. It may be due to vibration of the knife in respect of the specimen. In vitreous cryo-sections, it is generally due to variation of friction of the section on the knife surface.  Chemical mapping  Construction of 2– or 3–dimensional maps of the chemical composition of specimens for one or several elements.  Critical micelle concentration  The CMC corresponds to the minimum (CMC) concentration of detergent at which micelles form. The CMC is very sensitive to temperature and polarity of the medium.  Colloidal gold marker  Colloidal gold bound to antibody (fragments) via hydrophobic and electrostatic interactions.  Contouring  Manual drawing of contour lines in slices of a tomogram.  Contrast transfer function  Function associated to the electron magnetic lens and which describes the proportion of signal that is transferred by the imaging system for each spatial frequency. The CTF is an oscillating function that varies with defocus value and the spherical aberration of the objective lens.  Conventional  A useful term to designate anything except the present subject. In this book, conventional electron microscopy is noncryo-electron microscopy. More specifically, a conventional preparation method means chemical fixation and resin embedding.  Correlative light microscopy (LM)  Combination of observing and imaging and electron microscopy (EM) with light microscopy before detailed analysis at high resolution with electron microscopy.  One of the major cutting artifacts  Crevasse induced by cutting stress in vitreous samples.  A simple formula that defines the  Crowther criterion theoretical resolution of a tomogram only in terms of the total number of projection images and the tilt range, which by definition also defines the tilt increment.


Glossary

661

 Cryo-fixation or cryo-immobilization 







 Solidification of a biological specimen by cooling with the aim of minimal displacement of its components.  A liquid that boils at extremely low Cryogen temperature, e.g., liquid nitrogen (boiling point 196°C). Liquid nitrogen is a primary cryogen. Other cryogens, such as liquid ethane, are referred to as secondary cryogens because they are cooled by liquid nitrogen.  Pertaining to preservation and/or Cryogenic maintenance of molecules, cells or organisms at extremely low temperatures, typically in liquid nitrogen. Cryo-section according to Tokuyasu  Ultrathin thawed frozen section. Biological samples are weakly fixed with aldehydes, protected against freezing damage by infiltration with sucrose, frozen in liquid nitrogen and sectioned at 90°C to 120°C by cryo-ultramicrotomy (usually used for immunolabeling.  Production of frozen-hydrated sections Cryo-sectioning for observation in a cryo-electron microscope (see CEMOVIS).

D  Dehydration

 Freeze drying

 Freeze-substitution  Depth-of-field

 Detergent  Devitrification


 Free water from the fixed sample must be removed when water immiscible resins are used for embedding.  Dehydration of cryofixed samples through the sublimation of ice (lyophilisation). Starting temperature of procedure: 100°C.  Dehydration of cryofixed samples by using an organic solvent carried out below 90°C.  The distance in object space over which the objective focused on the specimen can provide adequate definition or clarity (Dictionary of Science and Technology, Academic Press, San Diego, CA, 1992).  Amphiphilic molecules used to solubilize membrane proteins.  Crystallization of water by warming a vitrified sample. For pure water, the devitrification temperature is ca. 135°C. It is higher when the solute concentration is high.

662  Dialysis  DOGS-NTA-Ni  Dose fractionation


 Mechanism allowing removal of some components from a solution.  Synthetic lipid formed by a hydrophobic domain and a hydrophilic domain containing chelated nickel.  The process of distributing the total electron dose that can be tolerated by a specimen between the individual projection images (cryo-electron tomography).

E  EELS

 EFTEM

 Elvanol/Mowiol  Energy filtered image  Epi-fluorescence microscope

 Epon  Epoxy resin

 ESI


 Among other structural information, the electron energy-loss spectrum contains chemical information about the specimen in the form of specific edges (with specific shape and position in energy) superimposed on a decreasing background.  Energy filtered transmission electron microscopy can be used for the measurement of the elemental composition of irradiated specimens. This analysis is possible by the use of an imaging energy filter integrated into the microscope column or by using a prism spectrometer below the final screen.  Semisolid embedding medium for light microscopy.  See ESI.  The illumination light of the object is using the same light path through the objective as the emission signal.  Epoxy resin.  Chemically: polyaryl esters of glycerol with terminal epoxy groups and hydroxyl groups spaced along the chain. With addition of cross-linking agents, they are converted into an inert solid.  Generates images with electrons of selected energy loss. These data concern only element-specific electrons = imaging for one element.

663

Glossary

 Etching

 Sublimation of a solvent for a short period of time.  In a liquid form, ethane is the ideal vitrification coolant.

 Ethane

F  Fiducial slang)

marker

(or

“fiducial,”  An exogenous, inorganic colloidal particle, usually 10 nm colloidal gold, introduced into or onto a specimen for the purposes of facilitating subsequent alignment of projection images.

 Fixation

 The goals of fixation are to preserve the structure of samples with minimum alterations from the living state.

 Fluorochrome

 A chemical component, which upon illumination radiates light at a longer wave length.

 Formaldehyde

 Weak fixative that has little influence on immunolabeling efficiency.

 Fourier space

 Synonymous of reciprocal space; the “space” defining the Fourier transform of an object, i.e., its decomposition into a continuous spectrum of its component frequencies, as opposed to Euclidean or “real” space, where positions are defined in terms of an x, y, z coordinate system.

 Freeze-drying

 A technique by which the frozen water of a cryo-fixed specimen is removed by sublimation at low temperature in a vacuum chamber.

 Freeze-etching

 After breaking the specimen and before evaporating metal, the specimen undergoes a process of etching, i.e., ice sublimation, for a short period of time.

 Freeze-fracturing

 To break a frozen specimen into pieces and immediately make a heavy metal replica of the fractured plane.


664  Freeze-substitution


 Dehydration method by replacing water (in solid state) for a (fluid) solvent (mostly methanol, ethanol or acetone). After freeze substitution, the sample can be warmed up to room temperature (i.e., the process to dehydrate and chemically fix specimens at temperatures between 90°C and 30°C).

G  Gaussian denoising

 Glow discharge

 Glutaraldehyde  Gold enhancement

 Gold toning

 Green fluorescent protein

 Denoising of a signal with the assumption that the noise has a Gaussian distribution, or in other words, that it is “white noise.”  Procedure by which ionized atoms are deposited onto a carbon support to modify its surface properties. The carbon-coated grids are deposited onto an electrode that is used to ionize residual gas under vacuum.  Aliphatic dialdehyde, an efficient crosslinking fixative. It may considerably reduce immunolabeling efficiency.  Similar to silver–enhancement. Small gold particles increase size by gold atoms, instead of silver atoms, deposited onto the surface.  Treatment of silver–enhanced structures with gold chloride. Originally used to improve contrast in LM and to cover/enlarge silver enhanced gold.  Protein from the jelly fish Aequorea victoria. GFP is a marker of gene expression and protein targeting. It is used for live cell imaging.

H  H+-ATPase

 HC-Pro


 An ion pump that actively transports hydrogen ions across lipid bilayers in exchange for ATP.  The helper component proteinase is encoded by plant virus of the genus

665

Glossary

Potyvirus. HC-Pro is involved in different steps of the viral cycle, aphid transmission, replication, and virus cell-to-cell movement and is a suppressor of posttranscriptional gene silencing.  High-pressure freezing

 Rapid cooling 204.8 MPa.

of

a

sample

at

I  Immunoadsorption  Indirect labeling

 Purification of antibodies by adsorption on the specific antigen often bound to a sepharose column.  The primary antibody is not coupled to a marker molecule. The secondary antibody, which is used to detect bound primary antibodies, is coupled to a marker molecule.

K  Kelvin

 Baron William Thomson Kelvin 18241907), British physicist and mathematician, devised the Kelvin (K) temperature scale (Dictionary of Science and Technology, Academic Press, San Diego, CA, 1992). The international standard unit of temperature where 0° Kelvin is equivalent to 273°C.

L  Lipid monolayer

 Layer of lipids formed at the interface buffer-air.

 Low-dose

 Microscope operation mode with a beam-deflection unit that allows the precise determination of the total exposure dose on the specimen during data collection. Typically between 10 to 20 electrons/Å2.


666


 Lowicryl resin

 A mixture of acrylate-metacrylate resins with very low viscosity at low temperatures (up to 80°C).

 Low-temperature embedding

 Embedding in methacrylates, typically Lowicryls, at temperatures (below 0°C) by UV irradiation.  London Resin, brand name for methacrylates.

 LR White, LR Gold

M  MAPs

 Marker

 Methyl cellulose  Microtubules

 Missing pyramid

 Missing wedge

 Molecular distillation drying


 Proteins that interact with microtubules in the cell, regulating their dynamic behavior or allowing them to build complex organelles, such as axonemes and centrosomes.  Necessary to visualize the antigenantibody reaction by light microscopy (fluorescent molecules and enzymes) or electron microscopy (usually gold particles).  Embedding medium for ultrathin Tokuyasu cryo-sections.  Polymers of the tubulin molecule associated with proteins (MAPs) in the cell, and involved in various functions, such as cell division through the mitotic spindle, vesicular traffic through the action of molecular motors, cell compartmentalization, or cell motility through the motion of cilia and flagella.  Term used to describe the region of Fourier space that remains unsampled in a dual-axis tilting experiment, defined by orthogonal tilt axes. By combining two perpendicular recorded tomograms of the same region, the missing wedge can be reduced to a missing pyramid.  Missing information in Fourier space, due to limited tilt angles. Results in anisotropic resolution in X,Y,Z.  A special method of freeze-drying.

Glossary

667

N  NANOGOLD marker  Neurospora crassa  Numerical aperture

 Commercially available gold compound that can be covalently bound to antibodies and other molecules.  A type of red bread mold of the phylum Ascomycota.  Measure for the resolution power of an objective:  NA = n × sin α  n = Refraction index of the medium between sample and objective lens  α = Half opening angle of the objective.  The correlation with the resolution d is:  d = 0.61λ/NA  λ = The wave length of the electromagnetic wave.

O  Optimized position

 Osmication

 Osmium tetroxide

 Theoretical positions in a perfectly adjusted specimen stage where the distance between the optical axis of the microscope and the tilt axis of the specimen is zero.  A method to stain and fix biological specimens with the vapors from osmium tetroxide crystals or with an osmium tetroxide solution.  Strong fixative, but also contrasting agent especially for membranes. It may degrade proteins and, therefore, has a strong effect on immunolabeling efficiency.

P  Phosphotungstic acid  Photobleaching


 Heavy metal chemical used for negative staining.  Loss of intensity of the fluorescence signal during imaging. Fluorochromes are affected by radical oxygen species produced upon illumination.

668  Primary antibody  Protective colloid  Protein A


 Antibody specific to the antigen/epitope under investigation (usually raised in rabbit or mouse).  Slows down silver enhancement and makes the reaction more efficient and reproducible.  In EM, often used instead of secondary antibody. Binds to a number of IgGs from different species.

Q  Quantum dot (QD) marker

 Fluorescent semiconductor nanocrystal covalently bound to antibodies. Most QDs exhibit relatively low electron density, but can be silver-enhanced.

R  Replica  Resin embedding

 Resin polymerization  Rotary shadowing

 The metal imprint or cast of the surface of an object.  Dry specimens are fragile and porous and must be permeated with a fluid resin that can polymerized resin for conservation and sectioning.  Resin can be polymerized by heat or by UV radiation with addition of accelerating agents: cross-linking and catalysts.  The specimen is tilted under a chosen angle and rotated while shadowing.

S  Secondary antibody

 Antibody (usually raised in goat) coupled to a marker molecule that detects the bound primary antibody.

 Segmentation

 The delineation of the features of a complex, three-dimensional image, either manually using a mouse or drawing tablet, or automatically by means of appropriate, e.g., edge-detection, algorithms.


Glossary

 Shadowing

 Silver enhancement

 Silver stabilization

 SIMS (SIMS imaging)

 Simultaneous iterative reconstruction technique (SIRT)  Slam-freezing

 Spurr’s resin


669  To evaporate a heavy metal from a point source at an oblique angle onto a specimen surface in order to produce a cast of the specimen.  Process similar to photographic development. In the presence of a reducing agent, the gold surface acts as a catalyst for the reduction of silver ions to metallic silver. The metallic silver deposits on the gold surface, resulting in a growing silver layer.  Protection of the silver layer that is sensitive to oxidation (OsO4, electron beam, air humidity), by gold chloride treatment.  SIMS is a mass spectrometric technique using a primary ion beam to analyze the specimen. Chemical and isotopic microanalyses can be carried out by focusing this primary beam to get a microprobe (diameter under 100 nm). SIMS imaging is obtained by scanning such a microprobe over the surface of interest of the specimen.  In TOF-SIMS, the primary ion beam is pulsed and a whole mass spectrum can be analyzed. This imaging method gives molecular information, but is less sensitive than dynamic SIMS imaging.  A reconstruction algorithm similar to ART (see above) where the reconstructed volume is updated only after all corrections have been performed.  Cryo-fixation technique where the surface of a biological sample is cooled down by the polished surface of a metal block, which, in turn, is cooled by the cryogen.  One of the most fluid resins used in electron microscopy (about 60 cP at ambient temperature for fresh mixture). Easily sectioned and resistant under the analytical probes.

670


T  Tomogram

 Tomography

 Torr

 Transmission fluid

 Triton X100

 Computed 3-D volume reconstruction of a specimen by using as many projection images as possible (usually between 121 and 141 images).  The process of obtaining a threedimensional image volume (tomogram) from a series of two-dimensional images, represented either by x-y slices, (e.g., confocal microscopy) or projections, (e.g., x-ray tomography or electron tomography).  Old unit for measuring vacuum still frequently used in electron microscopy. Derived from an Italian physicist and mathematician, Evangelista Torricelli (1608–1647), who invented the mercury barometer and was the first to create vacuum. 1 Torr = 1.33 × 102 Pa or N/m2 (SI unit for pressure).  A fluid used to mediate transfer of heat and pressure during high-pressure freezing without interacting with cellular specimens.  A nonionic surfactant that has a hydrophilic polyethylene oxide group and a hydrocarbon or hydrophobic group.

U  Uranyl acetate

 Fixative and contrasting agent. The divalent uranyl cation forms salt bridges with negatively charged groups, e.g., phosphate groups, hence, stabilizing membranes.

V  Vitreous


 From the Latin, literally “glass-like”, often used interchangeably with “amorphous.”

Glossary

 Vitrification

671  The formation of a non-crystalline (vitreous) solid state.

W  Weighted back-projection (WBP)

 Synonymous of filtered backprojection; the standard method of reprojecting aligned, two-dimensional projection images into a three-dimensional volume; the weighting factor is used to account for differences in low- and highfrequency information in Fourier space.

 Well-freezing/well-frozen

 Term to describe the state of solid water in a biological object. A sample is said to be well frozen if ice crystal ramifications formed cannot be seen with the electron microscope.

 Whole mount labeling

 Immunolabeling of large samples that have been permeabilized and extracted (e.g., by detergent or solvent treatment) prior to antibody and marker incubation to allow their penetration into the sample.


Handbook of Cryo-Preparation Methods for Electron Microscopy (Methods in Visualization)

Electron Microscopy Methods and Protocols

Electron Microscopy Methods and Protocols

Electron Microscopy Methods and Protocols

Electron Microscopy: Methods and Protocols

Methods in Plant Electron Microscopy and Cytochemistry

In Situ Hybridization in Light Microscopy (Methods in Visualization)

Topology-based Methods in Visualization

Topology-Based Methods in Visualization

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Electron Microscopy

Atomic Force Microscopy Methods

Microscopy, Immunohistochemistry, and Antigen Retrieval Methods: For Light and Electron Microscopy

Electron Microscopy: Methods and Protocols 2nd Edition (Methods in Molecular Biology Vol 369)

Electron tomography: methods for three-dimensional visualization of structures in the cell

Confocal Microscopy Methods and Protocols

Immunoelectron Microscopy: Methods and Protocols (Methods in Molecular Biology 657)

Light Microscopy: Methods and Protocols (Methods in Molecular Biology 689)

Electron Tomography: Methods for Three-Dimensional Visualization of Structures in the Cell

Confocal Microscopy Methods and Protocols

HandbookScanning Electron Microscopy

Scanning Auger Electron Microscopy

High-Resolution Electron Microscopy

Electron Microscopy, 2nd Edition

Electro Microscopy Methods and Protocols

Handbook of neuroprosthetic methods

Handbook of Tableau Methods

Topology-Based Methods in Visualization II

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Handbook of Proteomic Methods

Handbook of Neuroprosthetic Methods

Handbook of Cryo-Preparation Methods for Electron Microscopy (Methods in Visualization)

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Electron Microscopy Methods and Protocols

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Electron Microscopy: Methods and Protocols

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In Situ Hybridization in Light Microscopy (Methods in Visualization)

Topology-based Methods in Visualization

Topology-Based Methods in Visualization

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Microscopy, Immunohistochemistry, and Antigen Retrieval Methods: For Light and Electron Microscopy

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Immunoelectron Microscopy: Methods and Protocols (Methods in Molecular Biology 657)

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Electron Tomography: Methods for Three-Dimensional Visualization of Structures in the Cell

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