Advanced Techniques in Biological Electron Microscopy III

Edited by

J.K. Koehler

With Contributions by

A. P. Aguas M. F. Barbosa R. P. Bolender M. E. Cantino J. De Mey J. Frank D. E. Johnson M. Moeremans

K.-R. Peters P. Pinto da Silva M. Radermacher

With 110 Figures

Springer-Verlag Berlin Heidelberg New York Tokyo

JAMES K. KOEHLER, Ph.D. University of Washington

Department of Biological Structures School of Medicine

Seattle, W A 98195/USA

The cover illustration shows examples of chick fibroblasts labeled with antimyosin using fluorescence (a) and gold/silver techniques (b). The lower panels show a va­riety of cells and organelles viewed after the fracture label procedure using WGA-

ovomucoid-gold complex. See pages 263 and 222 respectively for details.

ISBN-13: 978-3-540-16400-5 e-ISBN-13: 978-3-642-71135-0 DOl: 10.1007/978-3-642-71135-0

Library of Congress Cataloging.in-Publication Data. (Revised for vol. 3). Koehler. James K.. 1933- . Ad· vanced techniques in biological electron microscopy. Includes bibliographies. Contents: 1. [Without special title] - v. 2. Specific ultrastructural probes - v. 3. [Without special title] 1. Electron microscopy- Technique­Collected works. I. Bullivant, S. II. Title. III. Title: Biological electron microscopy. QH212.E4K6 578'.45

72·91386

This work is subject to copyright. All rights are reserved, whether the whole or part of the material is con· cerned, specifically those of translation, reprinting, re·use of illustrations, broadcasting, reproduction by photo· copying machine or similar means, and storage in data banks. Under § 54 of the German Copyright Law, where copies are made for other than private use, a fee is payable to the publisher, the amount of the fee be

determined by agreement with the publisher.

© by Springer-Verlag Berlin Heidelberg 1986

So/leover reprint of the hardcover 1 st edition 1986

The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a spe· cific statement, that such names are exempt from the relevant protective laws and regulations and therefore

free for general use.

2131/3130-543210

Preface

This volume is a continuation of two prior books on advanced electron microscope techniques. The purpose of this series has been to provide in­depth analyses of methods which are considered to be at the leading edge of electron microscopic research procedures with applications in the biological sciences. The mission of the present volume remains that of a source book for the research practitioner or advanced student, especially one already well versed in basic electron optical methods. It is not meant to provide in­troductory material, nor can this modest volume hope to cover the entire spectrum of advanced technology now available in electron microscopy.

In the past decade, computers have found their way into many research laboratories thanks to the enormous increase in computing power and stor­age available at a modest cost. The ultrastructural area has also benefited from this expansion in a number of ways which will be illustrated in this volume. Half of the contributions discuss technologies that either directly or indirectly make extensive use of computer methods.

Biological stereology has long been an area requiring a high degree of training and expertise far beyond that of conventional descriptive electron mi­croscopy. Microcomputers have, however, provided the tool by which ul­trastructure labs not dedicated to stereology can utilize the technique to solve specific stereological problems. The missing link in this technology has been access to computer programs that can easily be adapted to individual stereological questions. Dr. BOLENDER not only outlines up to date stereo­logical methods, but discusses in some detail software packages available and their use in solving various stereological questions.

We are all aware of the dramatic results obtained by the use of electron diffraction methods on periodic structures such as viruses, nucleosomes and gap junctions. However, the use of such techniques to make structural de­terminations on nonperiodic materials has been slow to evolve. Drs. FRANKE and RADERMACHER discuss progress in this field and outline procedures which allow the reconstruction of three dimensional molecular information from microscopic data and illustrate these methods with several timely exam­ples.

Drs. JOHNSON and CANTINO describe the use of energy dispersive X-ray spectrometry with emphasis on the detection of diffusible ions. This area of fine structure research, particularly the quantitative aspects, was for many years an intractable subject, ripe for exploration but replete with pit-

VI Preface

falls, even for experienced microscopists. The basic procedures for obtaining spectral results are reviewed in this chapter as well as the background needed for preparing samples for analysis.

Replica and shadowing procedures are often overlooked as a preparative method by biologically oriented electron microscopists. Such methods, how­ever, are sometimes the simplest and most effective way of examining certain types of specimen, and account for a significant fraction of preparative work. This, together with recent advances in high resolution scanning microscopy, make Dr. PETER's critical evaluation of metal deposition procedures very timely. It may be surprising, even to experienced microscopists, that new and improved metal deposition technology is now available that can greatly im­prove or enhance resolution of replica and SEM preparations.

The use of colloidal gold as a marker for cytochemical EM localizations has grown enormously over the past few years. Unfortunately, the manner of this use is often not optimal or efficient, due to a lack of precise information concerning labeling parameters such as temperature, concentration. ligand characteristics, etc. Drs. DE MEY and MOEREMANS carefully outline the pre­parative steps to be followed and the application of these markers under vari­ous experimental situations. Topics often neglected in treatments such as the storage and purification of labeled material are thoroughly covered.

In the fracture-label technique discussed by Dr. PINTO DA SILVA et aI., we are taken a step beyond freeze-fracture methods and introduced to a pro­cedure by which we can investigate the nature and chemical make-up of the interiors of biological membranes. This unique combination of freeze-frac­ture, labeling and sectioning methods, as well as the interpretation and evaluation of the results, are described in full detail.

I would like to express my appreciation to this outstanding panel of re­searchers for their contributions, as well as to the staff of the Springer-Ver­lag, for their excellent editorial scrutiny and valuable suggestions in the pro­duction of this volume.

Seattle, Spring 1986 JAMES K. KOEHLER

Contents

Three-Dimensional Reconstruction of Nonperiodic Macromolecular Assemblies from Electron Micrographs

J. FRANK and M. RADERMACHER (With 30 Figures)

1 Introduction 1.1 General 1.2 The Three-Dimensional Structure as an Average

2 Methods for Obtaining Projection Data 2.1 What is Being Reconstructed? 2.2 Scanning of Projection Data 2.3 Hardware and Software for Electron Image Processing 2.4 Alignment of Projections . . . . . . . . . . 2.5 Methods for Obtaining Statistically Significant Projections

3 Methods of Three-Dimensional Reconstruction 3.1 Preliminaries ......... . 3.2 Some Existing Algorithms for Reconstruction

3.2.1 Real-Space Methods .... 3.2.2 Fourier Methods . . . .

3.3 Resolution of the Reconstructed Object 3.4 The Point Spread Function and the Effect of Angular

Limitations ......... . 3.5 Conical Tilting Geometry . . . . . 3.6 Experimental Data Collection Methods

3.6.1 Goniometer Tilting . . . . . 3.6.2 Use of Multiple A Priori Orientations 3.6.3 Random Tilts of a Long, Quasi-Cylindrical Particle 3.6.4 Random In-Plane Rotations of a Particle Having a Fixed

Orientation . . . . . . . . . 3.7 Representation of Three-Dimensional Data

4 Experimental Results . . . . . . . . . 4.1 Ribosome . . . . . . . . . 4.2 Oxygen-Carrying Proteins: Hemocyanins and Hemoglobins

of Invertebrates ... . 4.3 Chromatin ..... . 4.4 Survey of Published Results

1

1

2 5 5

10 11

14 17 20

20

22 22 25 26

27

30 33 33 34 36

37 40 44 44

49 51 56

VIII Contents

5 Conclusions .............. . Appendix: Some Important Definitions and Theorems A.l The Two-Dimensional Fourier Transform A.2 Convolution A.3 Resolution in the Fourier Domain A.4 Low-Pass Filtration A.5 Correlation Functions A.6 The Projection Theorem

References . . . . . . .

56 57 57 59 60 61 62 63 64

High Resolution Biological X-Ray Microanalysis of Diffusable Ions D. E. JOHNSON and M. E. CANTINO (With 6 Figures)

1 Introduction ..... 2 Principles of the Technique 3 Specimen Preparation

3.1 Introduction 3.2 Low Temperature Methods for AEM 3.3 Tissue Preparation 3.4 Rapid Freezing 3.5 Cryoultramicrotomy 3.6 Frozen Transfer 3.7 Freezing Drying

4 Quantitation 4.1 Theory of Quantitation of Biological Thin Samples 4.2 Errors in Quantitation ...... . 4.3 Mapping ........... .

5 Applications in the Analysis of Diffusab1e Ions 6 Conclusion References

Metal Deposition by High-Energy Sputtering for High Magnification Electron Microscopy

K-R. PETERS (With 32 Figures)

1 Introduction ...... . 1.1 Increase of Surface Information 1.2 Background . . . . . . .

1.2.1 Enhancement of Topographic Contrast 1.2.2 Increase of Contrast at High Magnification 1.2.3 High Resolution Replicas ..... .

73 73 75 75 76 78 81

83 85 85 88 88 90 93 95 96 97

101 101 102 102 103 103

Contents

l.2.4 Scanning Electron Microscopy l.2.5 Visualization of Surface Fine Structures

l.3 Contrast Principles ........ . l.4 Limiting Properties of Conventionally Used Metal Films

l.4.1 Metal Film Thickness 1.4.2 Particle Decoration

2 Methods ...... . 2.1 Microscopy .... .

2.l.1 Specimens and Preparation 2.l.2 Instruments 2.l.3 Useful Magnifications

2.2 Metal Deposition Technique 2.2.1 High Vacuum Metal Deposition System 2.2.2 Geometric Factors for Metal Deposition 2.2.3 Film Thickness Measurements

3 Metal Deposition ..... 3.1 Metal Film Formation

3.1.1 Phases of Film Growth 3.l.2 Nucleation .... 3.l.3 Film Growth from Nuclei

3.2 Low-Rate High-Energy Sputter Deposition 3.2.1 Energy Parameters 3.2.2 Low-Rate Deposition

3.3 Thin Continuous Film Production 3.3.1 Reduction of Decoration Effects 3.3.2 Reduction of Scattering 3.3.3 Reduction of Self-Shadowing 3.3.4 Reduction of Contamination 3.3.5 Critical Film Thickness

3.4 Coating Strategy .... 3.4.1 Surface Information 3.4.2 Rationales for Continuous Film Application 3.4.3 Choice of Coating Technique 3.4.4 Test Specimen

4 Conclusion References

Computer Programs for Biological Stereology R. P. BOLENDER (With 5 Figures)

Introduction ........ . 1.1 Stereology: An Overview . . . . 1.2 Biological Applications of Stereology

IX

105 107 III ll2 ll5 ll6 ll8 ll8 ll8 ll9 ll9 120 120 123 129 132 132 133 133 134 134 135 135 140 140 142 142 144 146 147 147 149 151 153 156 159

167 167 168

x Contents

2 Point Counting Programs .... 169 2.1 Point Counting Stereology: An Overview 169 2.2 Hardware ..... 174 2.3 Software ..... 176

2.3.1 Experiment Design 176 2.3.2 Data Input 177 2.3.3 Data Management 178 2.3.4 Data Analysis 178 2.3.5 Data Output 179

3 Digitizing Programs 180 3.1 Digitizing Stereology: An Overview 180 3.2 Hardware 185 3.3 Software 185

3.3.1 Data Collected 185 3.3.2 Data Analysis 186

4 Special Purpose Stereo logy Programs 186 4.1 Size Frequency Distributions and Mean Caliper Diameters 188

4.1.1 Convex Structures 189 4.1.2 Nonconvex Structures 191 4.1.3 Oriented Structures 191

4.2 Sampling Analysis 192 4.3 Quantification of Freeze-Fracture Replicas 192 4.4 Pattern Analysis 193

5 Concluding Comments 194 References ..... 195

A Guide to Fracture Label: Cytochemical Labeling of Freeze-Fractured Cells

P. PINTO DASILVA, M. 1. F. BARBOSA, and A. P. AGUAS (With 25 Figures)

1 Introduction ........... . 1.1 Freeze-Fracture: Membrane Splitting 1.2 Freeze Etching: Membrane Cytochemistry 1.3 Fracture Label: Freeze-Fracture Cytochemistry

2 Experimental Procedures 2.1 Main Steps 2.2 Preparation of Specimens

2.2.1 Fixation .... 2.2.2 Embedding in BSA 2.2.3 Impregnation. . .

201 201 202 203 204 204 204 204 204 207

Contents XI

2.2.4 Freezing 207

2.2.5 The Sandwich Method 207

2.3 Freeze Fracture . . . . . 207 2.3.1 Thin-Section Fracture Label (TS-FL) 207

2.3.2 Critical Point Drying Fracture Label (CPD-FL) 208

2.4 Thawing and Deglycerination . . . . . . . . . 208 2.4.1 Thin-Section Fracture Label (TS-FL) . . . . 208

2.4.2 Critical Point Drying Fracture Label (CPD-FL) 209 2.5 Labeling. . . . . . . . . . . . . . . . . 209

2.5.1 Detection of Concanavalin A Binding Sites. . 209

2.5.2 Detection of Wheat-Germ Agglutinin Binding Sites 210

2.6 Processing of Labeled Specimes for Electron Microscopy 210 2.6.1 Thin-Section Fracture Label 210

2.6.2 Critical Point Drying Fracture Label 210 3 Electron Microscopy ....... 211

3.1 Thin-Section Fracture Label 211 3.2 Critical Point Drying Fracture Label 213

4 Interpretation ......... 21 7 4.1 Postfracture Reorganization of Membrane Components 217

4.2 Labeling of Outer Surface Receptors on Protoplasmic Membrane Halves ............ 221

4.3 Labeling of Intracellular Membranes and Nuclear Matrix 222

4.4 Commentary 223 References 224

The Preparation of Colloidal Gold Probes and Their Use as Marker in Electron Microscopy

J. DE MEY and M. MOEREMANS (With 12 Figures)

Introduction .............. 229 2 The Preparation and Storage of Colloidal Gold Sols 231

2.1 Introductory Remarks ......... 231 2.2 Citrate Gold (15 nm). (Reducing Agent = Sodium Citrate) 233

2.2.1 Stock Solutions ........... 233

2.2.2 Procedure . . . . . . . . . . . 233

2.2.3 Variation of the Size by Using Different Citrate Concentrations ........... 234

2.3 A Modified Citrate Method for Producing 8-10 nm Gold 234

2.4 Phosphorous Gold (Reducing Agent = White Phosphorous) 235

2.4.1 Introductory Remarks 235

2.4.2 Stock Solutions 235 2.4.3 Procedure 235

XII Contents

2.5 The Alternative Citrate-Tannic Acid Procedure for Preparing Small Gold Particles . . . . . . . . 236 2.5.1 The Procedure of MULPFORDT . . . . . . 236 2.5.2 The Procedure of SLOT and GEUZE .... 236

3 The Production, Purification, and Storage of Gold Probes 237 3.1 General Remarks About the Adsorption of Proteins

to Colloidal Gold . . . . . . . . . . . . . 237 3.2 Preparation of Protein Solution and Colloidal Gold Sol

Before the Adsorption Step . . . . . . . . . . . 239 3.3 Determination of the Minimal Protecting Amount of Protein 239

3.3.1 Introductory Remarks . . . . . . . 239 3.3.2 Procedure ........... 240

3.4 Preparing, Purifying, and Storing a Gold Probe 241 3.4.1 General Remarks ........ 241 3.4.2 The Preparation of a Polyclonal Antibody/Gold Probe

for Use in Electron Microscopic Immunocytochemistry 242 3.4.3 The Preparation of a Monoclonal Antibody/Gold Probe

for Use in Electron Microscopic Immunocytochemistry 243 3.4.4 The Preparation of Streptavidin/Gold and Protein A/

Gold Probes for Use in Electron Microscopy 243 3.4.5 Use of Carbowax 20 M as Stabilizer . . . . 244

4 Quality Control and Analysis of Gold Sols and Probes 244 5 Critical Evaluation of the Use of Gold Probes in Selected

Marking Techniques ........ . 5.1 Influence of the Size of the Gold Particles

on Marking Efficiency ............. . 5.2 Gold Marking of Surface Components of Cells in Suspension

and Monolayers ...... . 5.3 EM Localization of Targets in Tissues

5.3.1 General Remarks .... 5.3.2 Immunomarking of Ultrathin Sections of Resin-

Embedded Tissues or Cells ...... . 5.3.3 Immunomarking of Thawed, Ultrathin Frozen Sections

of Tissues or Cells ............ . 5.3.4 Protein A/Gold or Secondary Antibody/Gold Probes? 5.3.5 Double on-Grid Marking ......... .

5.4 Pre-Embedding Marking of Intracellular Targets in Cultured Cell Monolayers

6 Conclusions References

Subject Index

247

247

248 251 251

252

256 259 260

260 265 266

273

Contributors

AGUAS, A. P., Section of Membrane Biology, Building 538, Room 104, NCI­FCRF, Frederick, MD 21701, USA

BARBOSA, M. F., Section of Membrane Biology, Building 538, Room 104, NCI-FCRF, Frederick, MD 21701, USA

BOLENDER, ROBERT P., University of Washington, Department of Biologi­cal Structure, Seattle, W A 98195, USA

CANTINO, MARIE E., University of Washington, Division of Bioengineering, School of Medicine, Seattle, W A 98195, USA

DE MEY, JAN, Laboratory of Biochemical Cytology, Janssen Pharmaceutica, Turnhoutsweg 30, 2340 Beerse, Belgium

FRANK, JOACHIM, State of New York, Department of Health, Tower Build­ing, Albany, NY 12201, USA

JOHNSON, DALE E., University of Washington, Division of Bioengineering, School of Medicine, Seattle, W A 98195, USA

MOEREMANS, MARC, Laboratory of Biochemical Cytology, Janssen Phar­maceutica, Turnhoutsweg 30, 2340 Beerse, Belgium

PETERS, KLAUS-RUDIGER, Yale University, Section of Cell Biology, School of Medicine, New Haven, CT 06510, USA

PINTO DA SILVA, PEDRO, Dept. of Health & Human Services, Building 538, Room 104, NCI-FCRF, Frederick, MD 21701, USA

RADERMACHER, M., State of New York, Department of Health, Empire State Plaza, Albany, NY 12201, USA