Introduction to electron microscopy for biologists /

Table Of Contents:
Contributors xiii

PART I Exploring the Organisation of the Cell by Electron Microscopy

Section 1 Basic Transmission and Scanning Electron Microscopy

High Pressure Freezing and Freeze Substitution of Schizosacharomyces pombe and Saccharomyces cerevisiae for TEM

Stephen Murray

Introduction 4(1)

Materials and Instrumentation 4(3)

Procedures 7(3)

Comments and Problems 10(10)

References 17(3)

Electron Probe X-ray Microanalysis for the Study of Cell Physiology

E. Fernandez-Segura

Alice Warley

Introduction 20(1)

Rationale 21(1)

Methods 22(15)

Equipment 37(1)

Discussion 38(8)

References 40(6)

Preparation of Cells and Tissues for Immuno EM

Paul Webster

Heinz Schwarz

Gareth Griffiths

Preparing Biological Specimens for Examination by Electron Microscopy 46(2)

Vitrification and Chemical Fixation for Immunolocalization 48(1)

Vitrification Followed by Freeze Substitution 48(1)

Chemical Cross-linking (or Fixation) 49(1)

Embedding in Resin for Sectioning 50(1)

Cryosectioning for Immunocytochemistry: The Tokuyasu Method 50(1)

The Starting Material 51(1)

Protocols 51(2)

Alternative Approaches to Freeze Substitution 53(1)

Correlative Microscopy 54(5)

References 55(4)

Quantification of Structures and Gold Labeling in Transmission Electron Microscopy

John Lucocq

Introduction 59(1)

Sampling and Stereology 60(2)

Quantities Displayed on Sections---Gold Labeling and Profile Data 62(7)

Quantities in Three Dimension 69(10)

Spatial Analysis 79(5)

References 80(4)

Combined Video Fluorescence and 3D Electron Microscopy

Alexander A. Mironov

Roman S. Polishchuk

Galina V. Beznoussenko

Introduction 84(1)

Rationale 84(1)

Method Steps 85(2)

Immunolabeling for EM with NANOGOLD 87(1)

Immunolabeling for EM with HRP 88(10)

References 95(3)

From Live-Cell Imaging to Scanning Electron Microscopy (SEM): The Use of Green Fluorescent Protein (GFP) as a Common Label

Sheona P. Drummond

Terence D. Allen

Introduction 98(1)

Rationale 99(1)

Methods 99(4)

Summary 103(1)

Concluding Remarks 104(6)

References 107(3)

Immunolabeling for Scanning Electron Microscopy (SEM) and Field Emission SEM

Martin W. Goldberg

Introduction 110(10)

Methods 120(2)

Procedure 122(10)

Conclusions 129(1)

References 129(3)

Immunogold Labeling of Thawed Cryosections

Peter J. Peters

Jason Pierson

Introduction 132(1)

Preparation of Carbon-and Formvar-Coated Copper Girds 132(2)

Aldehyde Fixation of Cells for Cryoimmuno Labeling 134(2)

Embedding Samples for Cryoimmuno Labeling 136(2)

Cryosectioning for Cryoimmuno Labeling 138(2)

Immunogold Labeling 140(3)

Reagents and Solutions 143(3)

Future Outlooks 146(6)

References 148(4)

Close-to-Native Ultrastructural Preservation by High Pressure Freezing

Dimitri Vanhecke

Werner Graber

Daniel Studer

Introduction 152(3)

Rational 155(1)

Material 156(2)

Methods 158(1)

High Pressure Frozen Samples 159(1)

Discussion 160(6)

References 162(4)

High-Pressure Freezing and Low-Temperature Fixation of Cell Monolayers Grown on Sapphire Coverslips

Siegfried Reipert

Gerhard Wiche

Introduction 166(1)

Materials and Instrumentation 167(1)

Procedures 167(7)

Comments and Pitfalls 174(8)

References 180(2)

Freeze-Fracture Cytochemistry in Cell Biology

Nicholas J. Severs

Horst Robenek

Introduction 182(1)

Basic Rationale: Solving the Problems of Combining Freeze Fracture with Cytochemistry 183(2)

Methods 185(6)

Discussion: Impact on Topical Questions in Cell Biology 191(10)

Concluding Comment 201(7)

References 202(6)

PART II Electron Microscopy of Specific Cellular Structure

Section 1 The Cell Membrane

Three-Dimensional Molecular Architecture of the Plasma-Membrane-Associated Cytoskeleton as Reconstructed by Freeze-Etch Electron Tomography

Nobuhiro Morone

Chieko Nakada

Yasuhiro Umemura

Jiro Usukura

Akihiro Kusumi

Introduction 208(4)

Protocol for Visualization of the Three-Dimensional Structure of the MSK of the Cytoplasmic Surface of the Plasma Membrane 212(7)

3D Structure of the Cytoskeleton-Plasma Membrane Interface 219(12)

Electron Tomography Clarified that Some of the Actin Filaments are Laterally Bound to the Cytoplasmic Surface of the Plasma Membrane 231(6)

References 232(5)

Visulization of Dynamins

Jason A. Mears

Jenny E. Hinshaw

Introduction 237(3)

Methods and Materials 240(11)

Discussion 251(2)

Summary 253(5)

References 254(4)

Section 2 The Cytoskeleton

Correlated Light and Electron Microscopy of the Cytoskeleton

Sonja Auinger

J. Victor Small

Introduction 258(1)

Materials and Methods 259(1)

Methods 260(6)

Results and Discussion 266(5)

Summary 271(3)

References 271(3)

Electron Microscopy of Intermediate Filaments: Teaming up with Atomic Force and Confocal Laser Scanning Microscopy

Laurent Kreplak

Karsten Richter

Ueli Aebi

Harald Hermann

Introduction 274(3)

Rationale 277(1)

Visualization of Intermediate Filaments in vitro and in Cultured Cells 278(15)

Materials 293(1)

Conclusions and Outlook 294(6)

References 295(5)

Studying Microtubules by Electron Microscopy

Carolyn Moores

Introduction 300(2)

Molecular Electron Microscopy 302(9)

Cellular Electron Microscopy 311(3)

Outlook 314(6)

References 315(5)

Section 3 Extracellular Matrix and Cell Junctions

Electron Microscopy of Collagen Fibril Structure In Vitro and In Vivo Including Three-Dimensional Reconstruction

Tobias Starborg

Yinhui Lu

Karl E. Kadler

David F. Holmes

Introduction 320(7)

Electron Microscopy of Isolated Collagen Fibrils 327(7)

Fibroblast/Fibril Interface in Developing Tendon 334(6)

Discussion 340(1)

Conclusion 341(7)

References 341(7)

Visualization of Desmosomes in the Electron Microscope

Anthea Scothern

David Garrod

Introduction 348(3)

Rationale 351(1)

Methods 351(9)

Materials 360(4)

Discussion 364(1)

Summary 364(4)

References 365(3)

Section 4 The Nucleus

A Protocol for Isolation and Visulization of Yeast Nuclei by Scanning Electron Microscopy

Stephen Murray

Elena Kiseleva

Introduction 368(2)

Materials and Instrumentation 370(2)

Procedures 372(13)

Comments and Problems 385(5)

References 386(4)

Scanning Electron Microscopy of Nuclear Structure

Terence D. Allen

Sandra A. Rutherford

Stephen Murray

Sheona P. Drummond

Martin W. Goldberg

Elena Kiseleva

Introduction 390(3)

Rationale 393(1)

Methods 393(7)

Colloidal Gold in the SEM 400(3)

Immunolabeling Protocol 403(1)

CPD for High Resolution SEM 404(3)

Discussion 407(5)

References 408(4)

Electron Microscopy of Lamin and the Nuclear Lamina in Caenorhabditis elegans

Merav Cohen

Rachel Santarella

Naama Wiesel

Iain Mattaj

Yosef Gruenbaum

General Introduction 412(2)

In Vitro Assembly of Ce-Lamin Filaments 414(2)

Preparation of Embryos and Adults for Transmission Electron Microscopy Using Microwave Fixation 416(3)

Preparation of C. elegans Embryos and Adults for Conventional Transmission Electron Microscopy by High Pressure Freezing Combined with Freeze Substitution 419(4)

Preembedding Immunogold EM Staining of Lamina Proteins in C. elegans Embryos 423(2)

Postembedding Immunogold EM Staining of Lamina Proteins in C. elegans Embryos 425(2)

Summary 427(5)

References 427(5)

Visualization of Nuclear Organization by Ultrastructural Cytochemistry

Macro Biggiogera

Stanislav Fakan

Introduction 432(1)

Cytochemical Contrasting Approaches 432(9)

High Resolution Autoradiography 441(1)

Immunocytochemistry 441(2)

Molecular in situ Hybridization 443(1)

Identification of Nucleic Acids by Means of Enzymatic Reactions 444(2)

Targeting of Intranuclear Substrates using Enzyme-Colloidal Gold Complexes 446(1)

Concluding Remarks 446(6)

References 447(5)

Scanning Electron Microscopy of Chromosomes

Gerhard Wanner

Elizabeth Schroeder-Reiter

Introduction 452(2)

Materials and Methods 454(2)

Chromosome Preparation 456(2)

Chromosome Structure in SEM 458(2)

Chromosome Analysis in SEM 460(10)

Outlook 470(7)

References 473(4)

PART III Cells and Infectious Agents

Infection at the Cellular Level

Christian Goosmann

Ulrike Abu Abed

Volker Brinkmann

Introduction 477(1)

Methods and Materials 478(16)

Overview and Conclusion 494(4)

References 495(3)

Electron Microscopy of Viruses and Virus-Cell Interactions

Peter Wild

Introduction 498(2)

Methods 500(15)

Material 515(1)

Discussion 516(4)

Summary 520(5)

References 521(4)
Index 525(12)
Volumes in Series 537