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more information – www.cambridge.org/9780521195997
Scanning Electron Microscopy for the Life Sciences

Recent developments in scanning electron microscopy (SEM) have resulted in a

wealth of new applications for cell and molecular biology, as well as related biological
disciplines. It is now possible to analyze macromolecular complexes within their
three-dimensional cellular microenvironment in near native states at high resolution,
and to identify specific molecules and their structural and molecular interactions. New
approaches include cryo-SEM applications and environmental SEM (ESEM), staining
techniques and processing applications combining embedding and resin-extraction for
imaging with high-resolution SEM, and advances in immuno-labeling. New develop-
ments include helium ion microscopy, automated block-face imaging combined with
serial sectioning inside an SEM chamber, and focused ion beam milling (FIB) combined
with block-face SEM. With chapters written by experts, this guide gives an overview of
SEM and sample processing for SEM, and highlights several advances in cell and
molecular biology that have greatly benefited from using conventional, cryo-,
immuno-, and high-resolution SEM.

Heide Schatten is Professor at the University of Missouri, Columbia. Her publications

include advanced imaging methods, cellular and molecular biology, cancer biology,
reproductive biology, microbiology, and space biology. The latter included collabora-
tions with NASA scientists and experiments aboard the Space Shuttle Endeavour to
examine the effects of spaceflight on cytoskeletal organization during development. She
has received numerous awards including grant awards from NASA, NIH, and NSF. She
has published over 200 papers and seven book chapters, and edited several special topics
journal issues and eight books, with several more in progress.
Advances in Microscopy and Microanalysis

Series Editors
Patricia Calarco, University of California, San Francisco
Michael Isaacson, University of California, Santa Cruz

Series Advisors
Bridget Carragher, The Scripps Research Institute
Wah Chiu, Baylor College of Medicine
Christian Colliex, Université Paris Sud
Ulrich Dahmen, Lawrence Berkeley National Laboratory
Mark Ellisman, University of California, San Diego
Peter Ingram, Duke University Medical Center
J. Richard McIntosh, University of Colorado
Giulio Pozzi, University of Bologna
John C. H. Spence, Arizona State University
Elmar Zeitler, Fritz-Haber Institute

Books in Series

Published
Heide Schatten, Scanning Electron Microscopy for the Life Sciences

Forthcoming
Nigel Browning et al., Dynamic Transmission Electron Microscopy
Michael Isaacson, Microscopic Nanocharacterization of Materials
Richard Leapman, Energy Filtered Electron Microscopy and Electron Spectroscopy
Scanning Electron Microscopy
for the Life Sciences
HEIDE SCHATTEN
University of Missouri
cambridge university press
Cambridge, New York, Melbourne, Madrid, Cape Town,
Singapore, São Paulo, Delhi, Mexico City
Cambridge University Press
The Edinburgh Building, Cambridge CB2 8RU, UK

Published in the United States of America by Cambridge University Press, New York

www.cambridge.org
Information on this title: www.cambridge.org/9780521195997

© Cambridge University Press 2013

This publication is in copyright. Subject to statutory exception

and to the provisions of relevant collective licensing agreements,
no reproduction of any part may take place without the written
permission of Cambridge University Press.

First published 2013

Printed and bound in the United Kingdom by the MPG Books Group

A catalog record for this publication is available from the British Library

Library of Congress Cataloging in Publication data

Schatten, Heide.
Scanning electron microscopy for the life sciences / Heide Schatten.
p. cm.
Includes index.
ISBN 978-0-521-19599-7
1. Biology – Methodology. 2. Scanning electron microscopy. I. Title.
QH324.9.X2S33 2013
570.280 25–dc23
2012015496

ISBN 978-0-521-19599-7 Hardback

Cambridge University Press has no responsibility for the persistence or

accuracy of URLs for external or third-party internet websites referred to
in this publication, and does not guarantee that any content on such
websites is, or will remain, accurate or appropriate.
 Contents

Endorsements page vii

List of contributors ix

1 The role of scanning electron microscopy in cell and molecular biology: SEM
basics, past accomplishments, and new frontiers 1
Heide Schatten

2 Corrosion casting technique 16

Jerzy Walocha, Jan A. Litwin, and Adam J. Miodoński

3 Revealing the internal structure of cells in three dimensions with scanning

electron microscopy 33
Sol Sepsenwol

4 Mitochondrial continuous intracellular network-structures visualized with

high-resolution field-emission scanning electron microscopy 50
T. Naguro, H. Nakane, and S. Inaga

5 Is the scanning mode the future of electron microscopy in cell biology? 71

Paul Walther, Christopher Schmid, Michaela Sailer, and Katharina Höhn

6 High-resolution labeling for correlative microscopy 83

Ralph M. Albrecht, Daryl A. Meyer, and O. E. Olorundare

7 The use of SEM to explore viral structure and trafficking 99

Jens M. Holl and Elizabeth R. Wright

8 High-resolution scanning electron microscopy of the nuclear surface in Herpes

Simplex Virus 1 infected cells 115
Peter Wild, Andres Kaech, and Miriam S. Lucas

9 Scanning electron microscopy of chromosomes: structural and analytical

investigations 137
Elizabeth Schroeder-Reiter and Gerhard Wanner

10 A method to visualize the microarchitecture of glycoprotein matrices with

scanning electron microscopy 165
Giuseppe Familiari, Rosemarie Heyn, Luciano Petruzziello, and Michela Relucenti
vi Contents

11 Scanning electron microscopy of cerebellar intrinsic circuits 179

Orlando J. Castejón

12 Application of in vivo cryotechnique to living animal organs examined by

scanning electron microscopy 196
Shinichi Ohno, Nobuo Terada, Nobuhiko Ohno, and Yasuhisa Fujii

13 SEM in dental research 211

Vladimir Dusevich, Jennifer R. Melander, and J. David Eick

14 SEM, teeth, and palaeoanthropology: the secret of ancient human diets 236
Alejandro Romero and Joaquín De Juan

Index 257

The color plates are to be found between pages 118 and 119.
Endorsements

“This book is an excellent exposition of the many-faceted field of biological scanning electron
microscopy. A brief introduction to the physics of SEM imaging is followed by an outstanding
selection of recent applications, which are written by leaders in their respective fields and which
include complete methodological details.”
Michael Marko, Wadsworth Center, New York State Department of Health

“This book, Scanning Electron Microscopy for the Life Sciences, edited and compiled by Dr. Heide
Schatten, comprises an extensive collection of articles demonstrating that the relevance of SEM to
biological research is of increasing importance. The book is a very valuable compendium for any
researcher interested in the fine structural morphology and chemistry of the cell and its compart-
ments, such as mitochondria, and the nucleus and its contents. This, combined with the wide array
of approaches, including recent ones, such as helium ion microscopy and block-face imaging
combined with serial sectioning inside the SEM chamber, are all covered in this well-illustrated and
also otherwise beautiful produced volume.”
Bert Menco, Northwestern University

“Scanning Electron Microscopy for the Life Sciences includes an outstanding array of chapters on
techniques for sample preparation and SEM imaging for a number of specimen types ranging from
entire organs to molecules. Chapters include detailed information on protocols for specimen
preparation and data collection, analysis, and presentation that have been applied to specific
biological systems. Even though the information presented is specific to the experimental systems
used in the laboratories of the authors, the methods and information provided will benefit all who
use SEM in their research.”
Robert Price, University of South Carolina School of Medicine

“The use of scanning electron microscopy in the life sciences has increased dramatically in the last
decade. This has given rise to advances in equipment and development of new techniques. This text
provides a needed survey of these advances; displaying the many ways that the beautiful three-
dimensional structural detail available with scanning electron microscopy can be exploited.
Scanning electron microscopy has long been prized for its ability to visualize high resolution
surface detail, but many of the chapters in this book also show how internal detail of cells and tissue
can be analyzed by scanning electron microscopy. This is an excellent introductory text for those
who want to incorporate scanning electron microscopy in their repertoire. However, the well-
written descriptions of cutting-edge techniques and the many ‘tips and tricks’ provided by expert
authors insure that even experienced scanning electron microscopists will find the book valuable.”
W. Gray Jerome, Vanderbilt University Medical Center
viii Endorsements

“This book is an excellent source of information about recent advances in the field of SEM for the
life sciences and will assist microscopists in gaining a greater depth of understanding.”
Cynthia S. Goldsmith, Centers for Disease Control and Prevention (CDC)

“Authoritative review of modern biological SEM methods. Advanced specimen preparation and
imaging methods reveal fine details not observable by other means.”
Charles Lyman, Lehigh University
Contributors

Ralph M. Albrecht
University of Wisconsin-Madison

Orlando J. Castejón
Universidad del Zulia, Venezuela

Joaquin De Juan
Universidad de Alicante, Spain

Vladimir Dusevich
University of Missouri – Kansas City

J. David Eick
University of Missouri – Kansas City

Giuseppe Familiari
Sapienza University of Rome, Italy

Yasuhisa Fujii
University of Yamanashi, Japan

Rosemarie Heyn
Sapienza University of Rome, Italy

Katharina Höhn
University of Heidelberg, Germany

Jens M. Holl
Emory University School of Medicine, Atlanta

S. Inaga
Tottori University, Japan
x List of contributors

Andres Kaech
University of Zürich

Jan A. Litwin
Jagiellonian University Medical College, Krakow, Poland

Miriam S. Lucas
Swiss Federal Institute of Technology, Zürich

Jennifer R. Melander
University of Missouri – Kansas City

Daryl A. Meyer
University of Wisconsin-Madison

Adam J. Miodonski
Jagiellonian University Medical College, Krakow, Poland

T. Naguro
Tottori University, Japan

H. Nakane
Tottori University, Japan

Shinichi Ohno
University of Yamanashi, Japan

Nobuhiko Ohno
University of Yamanashi, Japan

O. E. Olorundare
University of Wisconsin-Madison and University of Ilorin, Nigeria

Luciano Petruzziello
Sapienza University of Rome, Italy

Michela Relucenti
Sapienza University of Rome, Italy

Alejandro Romero
Universidad de Alicante, Spain

Michaela Sailer
Universität Ulm, Germany
 List of contributors xi

Heide Schatten
University of Missouri-Columbia

Christopher Schmid
Max-Planck-Institute for Molecular Physiology, Germany

Elizabeth Schroeder-Reiter
Ludwig-Maximilians-Universität München, Germany

Sol Sepsenwol
University of Wisconsin, Stevens Point

Nobuo Terada
University of Yamanashi, Japan

Jerzy Walocha
Jagiellonian University Medical College, Krakow

Paul Walther
Universität Ulm, Germany

Gerhard Wanner
Ludwig-Maximilians-Universität München, Germany

Peter Wild
Institute of Veterinary Anatomy and Virology, University of Zürich

Elizabeth R. Wright
Emory University School of Medicine, Atlanta
1 The role of scanning electron
microscopy in cell and molecular
biology: SEM basics, past
accomplishments, and new frontiers
Heide Schatten

1.1 Introduction

New developments in scanning electron microscopy (SEM) have resulted in a wealth

of new applications for cell and molecular biology as well as related biological
disciplines. New instrument developments coupled with new sample preparation
techniques have been key factors in the increasing popularity of this versatile research
tool. The desire to view biological material in native states at high resolution has
stimulated new approaches to cryo-SEM applications and environmental SEM
(ESEM). In addition, new staining techniques and novel processing applications
that combine embedding and resin-extraction for imaging with high-resolution
SEM has allowed new insights into structure–function relationships. Advances in
immuno-labeling have further enabled the identification of specific molecules and
their location within the cellular microenvironment. It is now possible to analyze
macromolecular complexes within their three-dimensional cellular environment in
near native states, and this in many cases has provided advantages over two-
dimensional imaging with transmission electron microscopy (TEM). New instrument
developments include helium ion microscopy that allows imaging greater details of
cellular components; new technology approaches include automated block-face
imaging combined with serial sectioning inside an SEM chamber that in recent
years has increasingly been utilized for a variety of biological applications. Focused
ion beam milling (FIB) combined with block-face SEM is among the newer
approaches to analyze cellular structure in three dimensions. The present chapter
introduces the basic features of SEM and sample processing for SEM, and highlights
several advances in cell and molecular biology that have greatly benefited from using
conventional, cryo-, immuno-, and high-resolution SEM.

Scanning Electron Microscopy for the Life Sciences, edited by H. Schatten. Published by Cambridge
University Press © Cambridge University Press 2012
2 Heide Schatten

1.2 The SEM as a versatile instrument for biological applications

As has been highlighted in previous review papers and books, the SEM is known for
its versatility, allowing imaging and analysis of large and small sample sizes and of a
diversity of specimens in multiple biological disciplines. Numerous articles are
available on SEM instrumentation, modes of operation, imaging capabilities, and
resolution (reviewed in Pawley, 2008; Schatten, 2008, 2011); new books are also
available that have addressed different aspects of SEM utilization (Schatten and
Pawley, 2008; several others are reviewed by Hawkes, 2009). In addition, recent
special topics issues of microscopy journals focused on SEM have been devoted to
specific biological and material science applications, demonstrating the increased
need for more specific information for the increased number of researchers applying
SEM to biomedicine and the basic sciences. In this section, the SEM is briefly
introduced and the importance of sample preparation for biomedicine and biology
is highlighted and detailed for routine sample preparation as well as for several
specific applications. Examples of sample preparations that have been designed for
specific cellular and molecular investigations are presented in the individual chapters
of this book.
For general information a schematic diagram, Figure 1.1, displays the basic compo-
nents of a conventional SEM.
Images in the SEM are generated by probing the specimen with a focused high-energy
beam of electrons that is scanned across the specimen in a raster scan pattern. The
electron beam interacts with the specimen surface, and interaction of the beam electrons
with the sample atoms produces signals that contain information about the specimen’s
surface topography and characteristic features. However, internal cellular structures can
also be visualized by using preparation methods that “peel” off the regular surface layers
and turn internal structures into surfaces that can then be viewed with SEM providing
information on surface and internal structures of intracellular components. In addition,
isolated cellular components can be visualized clearly by SEM. Such applications are
included in Section 1.3 and are detailed for specific applications in several chapters of
this book.
The incident electron beam interacting with the specimen produces emission of
low-energy (<50 eV) secondary electrons (SE), back-scattered electrons (BSEs), light
emission (cathodoluminescence), characteristic X-ray emission, specimen current, and
transmitted electrons and others as displayed in Figure 1.2 (color plate). For routine
SEM imaging an electron gun with a tungsten filament cathode or a lanthanum
hexaboride (LaB6) cathode is used while a field emission gun (FEG) is used for more
detailed SEM imaging (reviewed by Pawley, 2008; Schatten, 2008, 2011). Specific
detectors are used to generate information from the specimen: typically an Everhart–
Thornley detector is used for SEs, a type of scintillation-photomultiplier system, while
a dedicated detector of either a scintillation or semiconductor type is used for BSE
detection. For routine SEM imaging a secondary electron detector is used for conven-
tional imaging. This imaging mode may allow significant advantages over TEM, as the
 SEM in cell and molecular biology 3

Figure 1.1 The basic components of a conventional SEM.

Figure 1.2 The incident electron beam interacting with the specimen produces emission of low-energy
secondary electrons (SE), back-scattered electrons (BSEs), light emission (cathodoluminescence),
characteristic X-ray emission, specimen current and transmitted electrons, and others as shown.
(See plate section for color version.)
4 Heide Schatten

depth of field generates images that can readily be interpreted by the brain as three-
dimensional representation. BSEs are beam electrons that are reflected from the sample
by elastic scattering. The BSE signal intensity is related to the atomic number of the
specimen and can therefore provide information about the different elements contained
in the sample, which is oftentimes applied for imaging colloidal gold immunolabels of
c. 5–10 nm in diameter. While characteristic X-rays for elemental analysis are used for
biological applications, this form of analytical imaging is currently more frequently
utilized in the material sciences to identify the composition of elements in a sample.
However, new developments are in progress that may be amenable to biological
applications and find new utilization in biology (Newbury, 2008). In this book,
elemental analysis is described for the characterization of dental material (Dusevich
et al., Chapter 13 of this book). Characteristic X-rays are emitted when an inner shell
electron is removed from the sample by beam interaction, which causes a higher energy
electron to fill the inner shell and release characteristic energy.
As discussed in detail in several chapters of this book, newer variations of SEMs
include the ESEM that allows imaging of relatively unprocessed samples contained in
low vacuum or gas. While this mode of imaging is not entirely practical for all biological
samples it is excellent for biomaterials and several other biological samples as demon-
strated by Dusevich et al. (Chapter 13 of this book). Most samples viewed in conven-
tional SEM do require processing, which routinely includes chemical fixation with
glutaraldehyde or formaldehyde to stabilize the specimen’s mobile macromolecular
structure by chemical cross-linking of proteins and osmium tetroxide to stabilize lipids.
Cryofixation is being used to preserve structures in their close to native states, which can
be achieved with liquid nitrogen or liquid helium temperatures, as described below and
detailed in several chapters in this book.
For chemically fixed samples, dehydration follows to replace water with organic
solvents such as ethanol (or acetone) in incremental steps that gradually include increased
ratios of alcohol (or acetone) to water up to 100% alcohol steps. It is critically important
to dehydrate samples fully without leaving water residues to avoid sample preparation
artifacts. The preferred choice for sample drying is the critical point procedure, but there
are alternatives if a critical point dryer is not available. The dried sample is then mounted
on a specimen holder (also called specimen stub). A last step before sample analysis with
SEM includes conductive coating of the sample to prevent accumulation of static electric
fields which may be caused by electron irradiation during imaging.

1.3 General sample preparation for SEM

The four steps for sample preparation include a) fixation, b) dehydration, c) critical point
drying, and d) coating. All four steps can vary significantly and require modifications for
specific applications. Adequate sample preparation is critically important to maintain
structural integrity and obtain reliable information on cellular components and molecular
composition. Poor and inadequate specimen preparation undoubtedly causes artifacts
and may yield wrong information, which in some cases has caused confusion and serious
 SEM in cell and molecular biology 5

concern in the literature (reviewed by Heuser, 2003). A great variety of sample prepa-
ration techniques and methods is available, and these have been elaborated by various
investigators for specific research questions, some of which are presented in specific
chapters of this book and others have been reviewed recently (Schatten, 2011). For
routine SEM applications, the most common specimen preparation techniques are dis-
cussed below. Specialized sample preparation techniques are discussed in several of the
other chapters of this book.

1.3.1 Conventional sample preparation

A most commonly used protocol includes fixing the biological sample with aldehydes
such as 2% paraformaldehyde/2% glutaraldehyde in 0.1 M phosphate buffer (PBS)
followed by PBS rinses, post-fixation in 1% osmium tetroxide in 0.1% PBS, rinses in
PBS, dehydration in increasing series of ethanol or acetone, critical point drying,
mounting on aluminum stubs, and coating (reviewed in Schatten, 2011). The times for
the specific protocol steps vary, depending on sample size and sample characteristics. For
optimal results the investigator is referred to research papers in the specific area of
interest or to specific Methods books series such as Methods in Cell Biology that contain
detailed recipes with valuable notes sections addressing potential problems, hints, alter-
native approaches, and other most valuable information shared by researchers in their
fields. General processing information for SEM is given below.
Chemical fixation is most frequently used for SEM sample preparation, but chemical
fixation may destroy the structural integrity of certain structures or molecules of interest.
For example, because glutaraldehyde cross-links the free amino groups of polypeptides
and amino acids and inactivates enzymes it is inadequate for enzyme localization and
related studies. In this case and several others, alternative preparation methods may yield
better and more reliable results.
Dehydration is necessary to prepare samples for viewing in the SEM vacuum. As
mentioned above for fixation, dehydration may be damaging to biological structures, as
the shape of a macromolecule or membrane is produced and maintained by its inter-
actions with water. This interaction may be destroyed by removing water during the
dehydration process. Shrinkage of biological material has been observed and may need to
be calculated for accurate measurements after preparing soft tissue for SEM (Boyde and
Maconnachie, 1979, 1981). However, large macromolecules and their associated or
covalently attached structural components are preserved and not affected by dehydration
(Ris, 1985, 1988, 1990, 1991).
Critical point drying (CPD) is a most reliable method for drying samples in prepa-
ration for viewing in the SEM vacuum. However, several precautions are important to
avoid artifacts (Ris, 1985), as traces of water contaminating the intermediate liquid
used for drying (ethanol or acetone) or in the liquid CO2 transition fluid may distort
ultrastructure and induce considerable artifacts (Ris, 1985). Artifacts introduced by
residual water after CPD had caused historic debates when new structures termed
microtrabeculae (Porter and Stearns, 1981) were clearly identified as preparation
artifacts (Ris, 1985; reviewed in Heuser, 2003). These studies and numerous others
6 Heide Schatten

have highlighted the importance of proper sample preparation as well as critical

evaluation of instrumentation and accessory equipment. It further underlines the
importance of critical sample evaluation and data interpretation. Thorough CPD is
important for producing artifact-free specimens.
Coating of a specimen is an important aspect in sample preparation, as most biological
structures have insulating characteristics and need to be made more conductive by
applying a thin layer of metal to reduce the effects of charging (sample glaring; discussed
in Hermann and Müller, 1991; Hermann et al., 1996). Heavy metals or heavy metal
compounds are used as coating materials and include gold, gold/palladium, platinum,
tungsten, graphite, and others (Walther, 2008) that are deposited either by high-vacuum
evaporation or by low-vacuum sputter coating of the sample. Sample conductivity may
also be increased by using the OTO (osmium, thiocarbohydrazide, osmium) staining
method (Seligman et al., 1966; Malick et al., 1975; Familiari et al., Chapter 10 of this
book). Determining the correct amount of coating that eliminates charging but allows
reliable viewing of biological structures without obscuring the areas of interest is
critically important.
Coating also improves contrast; coating thickness may vary with different samples
(reviewed in Schatten, 2011).
Optimizing the coating thickness and applying specialized coating techniques
(Walther, 2008) may be required for delicate biological structures, especially when
viewing with low-voltage field emission SEM (LVFESEM) for which coating with
1–2 nm gold, palladium, or platinum applied by sputter-coating has been optimal for a
large variety of samples (reviewed in Schatten, 2008). Coating may affect biologically
relevant resolution (reviewed in Pawley, 2008). If charging problems are still encoun-
tered, alternative coating methods may need to be considered such as those developed by
Walther and Hentschel (1989), who introduced a double coating technique by which the
sample is first coated with a thin layer of heavy metal (platinum–carbon or tungsten with
an average thickness of 2–3 nm) followed by a 5–10 nm carbon layer (Walther, 2008).
Specific coating procedures have been described and discussed in the literature (Peters,
1980; 1982; 1985; 1986a; 1986; 1988; Peters and Fox, 1990; Walther, 2008; reviewed in
Schatten, 2008), which includes Pt, W, and Ta by DC-ion sputtering to view cells
growing on EM grids (Lindroth et al., 1988; Bell et al., 1989; Lindroth and Sundgren,
1989).
For optimal analysis of cellular and molecular components, appropriate sample prep-
aration and the investigator’s expertise with specific biological samples are among the
most important criteria for reliable results using SEM. More difficult preparations are
oftentimes encountered with plant samples or microbiology specimens including bacteria
or parasites for which new preparation techniques may need to be developed. An
example for the development of such techniques has been described for Toxoplasma
(Schatten and Ris, 2002, 2004; Schatten et al., 2003). In Toxoplasma, actin visualization
had been a problem and several hurdles had to be overcome through step-wise and
complementary approaches to determine localization of the fixation-sensitive actin-like
fibers. Dobrowolski et al. (1997) had used cryo-fixation to determine immunolocaliza-
tion of actin molecules, which established the presence of actin immunogenicity
 SEM in cell and molecular biology 7

underneath the Toxoplasma surface. Subsequent experiments were performed peeling off
the outer surface layer by quick treatment with detergent followed by cytoskeletal
stabilization and fixation that revealed actin-like fibers underneath the surface
(Schatten et al., 2003). This example demonstrates that specific biological expertise is
important to reveal structure–function relationships using SEM; new preparation meth-
ods may need to be designed paying attention to the specific biological characteristics and
dynamics that require specific biological knowledge to preserve delicate structures that
may respond differently to different chemicals used in the preparation protocols. The
importance of biological expertise to obtain optimal information is demonstrated in
numerous examples. The different requirements to preserve different structures reliably
had already been recognized in the early pioneer days of electron microscopy when most
samples were fixed in the cold, unknowingly destroying the cold-sensitive microtubule
fibers. Correlative microscopy (also see Albrecht et al., Chapter 6 of this book) is
oftentimes needed to obtain accurate information for biological material. When it was
recognized that cold-fixation indeed destroyed microtubules and the debate was settled,
other debates emerged that questioned the characteristics of microtubules that in cross-
sectioned EM samples were featured as short stubs, while immunofluorescence micro-
scopy with anti-tubulin antibodies revealed long microtubules that had not previously
been shown with TEM. These historic examples clearly show that sample preparation
and interpretation of results are highly important and may require several approaches for
reliable identification of biological material.
For plant material, optimal SEM preparation techniques are still being elaborated, as
plant cells are more difficult to prepare for SEM because of tissue rigidity resulting from
polysaccharide-containing cell walls and the large vacuole spaces within cells (Cox et al.,
2008). In several cases, use of protoplasts has been the choice for plant material, as
protoplasts can be analyzed after removal of the cell wall containing large amounts of
cellulose that hinders optimal processing. Isolation and detergent extractions of plant
material have resulted in stunning data for cellular components, as seen in Chapter 9 of
this book by Schroeder-Reiter and Wanner, which displays details of plant chromatin.
Specific processing for microorganisms has been described in excellent detail by
Erlandsen (2008).

1.3.2 Freezing methods

Ultra-rapid freezing is frequently used to preserve molecules in a more native state compared
to chemical fixation. Ultra-rapid freezing demands avoiding the formation of damaging ice
crystals in cells or tissue, which is accomplished by freezing at a rate of 104–105 degrees
C/second. At this cooling rate, cellular water becomes vitrified rather than forming ice
crystals. Several freezing methods are readily available, some of which are described in the
specific chapters of this book. The basic freezing methods include the following:
Plunge freezing allows an average depth of vitrification of c. 1–2 µm with minimal ice
crystal artifacts, which is achieved by plunging a specimen into a liquid cryogen
such as supercooled liquid nitrogen or supercooled ethane or propane.
8 Heide Schatten

Slam freezing (cold metal block freezing) allows an average depth of vitrification of
c. 10–15 µm with minimal ice crystal artifacts, which is achieved by slamming a
specimen onto a copper or silver block that has been chilled to −196 to −269 °C
with liquid nitrogen or liquid helium.
Propane jet freezing allows an average depth of vitrification of c. 40 µm with minimal
ice crystal artifacts, which is achieved by sandwiching a 200–500 µm thick speci-
men between two metal plates that are clamped into a device that directs jets of
liquid propane cooled with liquid nitrogen against both sides of the specimen
plates.
High-pressure freezing allows an average depth of vitrification of c. 500 µm with
minimal ice crystal artifacts. This modification of propane jet freezing or liquid
nitrogen freezing is achieved by pressurizing the specimen to 2100 atmospheres to
suppress or reduce growth of ice crystals at the moderate freezing rates that can be
achieved in the depth of the sample. High pressure lowers the freezing point of
water as well as the rate of ice crystal formation.
These basic freezing methods and several modifications have been applied with great
success to a variety of specimens (reviewed in Schatten, 2011). Freezing followed by
freeze drying is among the methods of choice for many applications in cell biology
(Pawley and Ris, 1987) and freezing followed by freeze-substitution has gained increas-
ing popularity (Erlandsen, 2008) for the superior ultrastructural preservation of cellular
components and structures. Direct observation of frozen specimens (Pawley et al., 1991)
has provided resolution above 3 nm, and freeze–fracture (Haggis and Pawley, 1988) has
been applied successfully to visualize and analyze intracellular structures (reviewed in
Pawley, 2008). Analysis of intracellular structure has further been accomplished by dry
fracture of tissue culture cells achieved by touching intact cells to the surface of adhesive
tape (Lim et al., 1987; Ris, 1988, 1989; Ris and Pawley, 1989; Sepsenwol, Chapter 3 of
this book), allowing excellent insights into intracellular structure. Viewing of incorpo-
rated labels that decorate internal cell structure can also be accomplished with this
method.
Among the advantages of cryofixation over chemical fixation is the arrest of cells in a
“life-like” state; cryo-immobilization takes only milliseconds compared to chemical
fixation, which may take seconds, or even longer. Freeze-substitution in acetone,
methanol, or other solvents is frequently used for subsequent processing and permanent
fixation.
In addition to freezing alone, several combination methods have also been used for
specific biological applications including cryo-SEM of chemically fixed cells, as
described by Erlandsen (2008). Other investigators have used chemical fixation with
very low concentrations of glutaraldehyde (0.1–1.0%) for 10 to 15 min to stabilize
macromolecules prior to cryo-immobilization (Centonze and Chen, 1995; Chen et al.,
1995). Such fixation approaches preserved macromolecular complexes excellently and
revealed remarkable detail of actin filaments (Erlandsen, 2008) after coating with
chromium, allowing clear visualization of the helical twists of two polypeptide chains
in the filament and 5 nm subunits (reviewed in Schatten, 2011). In other studies using
 SEM in cell and molecular biology 9

cryo-methods, biological resolution of 2–3 nm could be achieved when viewing macro-

molecular complexes with high-resolution in-lens cryo-SEM (Erlandsen et al., 2001),
revealing details of the glycocalyx on the extracellular surface of human platelets that
were labeled with three colloidal-gold markers to detect all three cell-adhesion molecules
in the glycocalyx. This study used plunge-freezing into propane chilled with liquid
nitrogen followed by partial freeze-drying at −85 °C and the double-coating method
developed by Walther et al. (1995). This coating method involves cryo-coating by
evaporation of 2 nm TaW at 45° through electron-beam deposition and 7–10 nm carbon
at 90 °C. The double-coating technique has also been used in numerous other applica-
tions including double-layer coating of yeast cells after high-pressure freezing and
freeze–fracture (reviewed in Erlandsen, 2008). All these examples clearly demonstrate
that combination methods of various complexities and high-resolution SEM coupled
with specific expertise of biological structure can be superior over other imaging methods
and can reveal unique three-dimensional information.
If an SEM is equipped with a cold stage for cryo-microscopy, additional preparation
techniques are available including cryo-fracture under vacuum, sputter coating and transfer
to the SEM cryo-stage while still frozen. This method is particularly useful for imaging and
analysis of temperature-sensitive cellular components including fats. Direct viewing of
frozen specimens in the SEM by using a cold stage had become possible when side-entry
eucentric goniometer stages were developed that could accept high-stability, cryotransfer
stage rods. Uncoated and slightly coated specimens have been viewed in this mode (Herter,
1991; Pawley et al., 1991; Müller et al., 1992; Boyde, 2008; Walther, 2008), allowing
detailed three-dimensional information to be obtained, as for example by stereo-imaging of
frozen-hydrated mitochondria. Combination methods include freeze–fracture followed by
the thaw–fix technique developed by Haggis (Haggis, 1987; Haggis and Pawley, 1988),
involving fresh-freezing in propane, freeze–fracture and thawing into fixative, critical point
drying and ion-beam-sputter coating with Pt before imaging. These examples also dem-
onstrate the usefulness of stereo-imaging that allows better understanding of complex
structural interactions of cellular components (reviewed in Pawley, 2008).

1.4 High-resolution low-voltage SEM and combination methods

Several chapters in this book describe use of high-resolution low-voltage field emission
SEM (HRLVFESEM) with great success to view and analyze isolated structures or delicate
internal cellular components. These applications have greatly benefited from the develop-
ment of field-emission sources that has allowed formation of an intense beam of low-
voltage electrons with small beam diameter (reviewed in Albrecht and Meyer, 2008).
HRLVFESEM has increasingly found new applications in cell and molecular biology
for the study of structure–function relationships on three-dimensional levels. In addition,
HRLVFESEM has also been an indispensable approach to image and analyze isolated
structures that previously had been analyzed mainly by TEM negative staining. These new
applications utilizing low-voltage electron microscopy take advantage of accelerating
voltages at or below 5 keV. With these new capabilities combined with improved sample
10 Heide Schatten

processing and sample coating as described in several chapters of this book, modern SEMs
can achieve resolution for biological material down to 2–5 nm, a level previously only
possible with TEM (reviewed in Schatten, 2011). As shown in subsequent chapters in this
book, detailed analysis of chromosomes, cytoskeletal components, viruses, and other
biological material has been performed using HRLVFESEM and revealed new detailed
information in three dimensions that is superior to data obtained with TEM.
New applications with cryo-SEM including cryo-microtomy of cryo-immobilized plant
and animal cells (Nusse and Van Aelsi, 1999; Walther and Müller, 1999) have yielded new
information on internal cellular structures. In these specific applications the surface of the
tissue block is examined rather than sections by cryo-SEM. Furthermore, a cryo-dual beam
instrument has been utilized that incorporates both focusing electrons (SEM) and focusing
ion beam (FIB) columns. Such applications have been employed by Mulders (2003) to
analyze biological samples including yeast, bacteria, and gut epithelial cells.
New combination methods have been developed that will be addressed below. These
new applications and others offer new approaches to identify biological components
reliably inside cells, as described in excellent detail in several chapters of this book. A
variety of different methods may need to be employed for optimal information, taking
into consideration that some biological specimens are more fragile and complex than
others, requiring more complex specimen preparation and processing.
In the author’s lab, FESEM has been used to image delicate mitotic spindles and sperm
asters yielding new information on cytoskeletal interactions in three dimensions (reviewed
in Pawley, 2008; Schatten, 2008, 2011). In addition, the technique has allowed analysis of
isolated centrosomes in three-dimensional configuration (Thompson-Coffe et al., 1996).

1.5 New developments and future perspectives

One of the goals for new instrument and sample preparation development is to achieve
higher resolution and imaging of samples in more native states. Such approaches have
been pursued in recent years by Boyde (2008) and by researchers designing various types
of microfluidic chamber that can be placed inside an SEM (Thiberge et al., 2004; Boyde,
2008). The design and testing of microfluidic chambers is intensively being pursued by
several groups with applications for transmission EM and SEM (Thiberge et al., 2004;
Klein et al., 2011).
A most impressive development first presented by Denk and Horstmann (2004)
introduced automated block face imaging combined with serial sectioning inside the
chamber of an SEM. This development required several technical modifications that have
been described in detail (Denk and Horstmann, 2004). With this new technology devel-
opment the authors were able to trace even the thinnest axons to identify synapses in
nerve tissue. The authors reported several hundred sections of 50–70 nm thickness that
will lead to further developments to reconstruct large areas of neuronal tissue. Building
on these developments, Knott et al. employed combination methods using light and
electron microscopy. Serial section SEM of adult brain tissue using focused ion beam
milling allowed visualization of the ultrastructure (Knott et al., 2008). Several
 SEM in cell and molecular biology 11

combination methods for live cell analysis using light microscopy followed by analysis
with SEM have further been employed for nerve cells (Knott et al., 2009). These studies
included identification of live structures of interest with confocal microscopy followed
by analysis with focused ion beam/scanning electron microscopy (FIB/SEM) and serial
block face/scanning electron microscopy (SBF/SEM). Such studies and others offer new
approaches aimed at studying live-like events at high resolution, and are currently being
pursued with predicted success (Denk et al., 2012). When coupled with new method
developments, such as the recently reported novel genetically encoded tag for correlated
light and electron microscopy (Shu et al., 2011), it can further be predicted that we have
entered a new area of discoveries on ultrastructural/functional levels that will have been
made possible as a result of these new instrument/method developments.
Among the newest instrument developments is scanning helium ion microscopy
(SHIM or HeIM), offering new advantages over conventional SEM (reviewed in
Morgan et al., 2006) including higher resolution and increased brightness. The high
source brightness and short wavelength of the helium ions yield qualitative data that
provide sharp images on a wide range of materials. As the secondary electron yield is
quite high, it allows for imaging with currents as low as 1 femtoamp. A surface resolution
of 0.24 nm has been demonstrated. The detectors provide information-rich images that
reveal topographic, material, crystallographic, and electrical properties of the sample. In
contrast to other ion beams, there is no discernible sample damage due to the relatively
low mass of the helium ion. Since 2007 this technology has been commercialized and
instruments have been utilized successfully.
Taken altogether, since its introduction as a research tool for the life sciences (reviewed
by Pawley, 2008), the SEM has enjoyed enormous utilization in cell and molecular
biology, facilitated by new sample preparation methods, advances in instrument develop-
ment and new technological approaches that have generated novel two- and three-
dimensional information for a variety of biological specimens. All chapters in this
book are written by scientists with specific expertise in their respective fields of science
and specific expertise in SEM methodology that can be applied to various other areas of
interest in cell and molecular biology.

1.6 Acknowledgments

Donald Connor’s professional help with the illustrations and Howard A. Wilson’s pro-
fessional help with the presentation of several images in the book are gratefully
acknowledged.

1.7 References

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12 Heide Schatten

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Erlandsen, S. L. et al. (2001). High resolution cryo-FESEM and detection of individual cell
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 SEM in cell and molecular biology 15

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2 Corrosion casting technique
Jerzy Walocha, Jan A. Litwin, and Adam J. Miodoński

2.1 Introduction

Among various techniques of biological material preparation for scanning electron

microscopy, corrosion casting – sometimes also referred to as microcorrosion casting –
is the method of choice for studying three-dimensional topography of microvascular
systems, offering spatial continuity of the cast vessels combined with high resolution and
quasi-3-D quality of SEM images (Figure 2.1). In essence, the technique requires
injection of the studied microvasculature with solidifiable medium and subsequent
corrosion (maceration) of the tissue. The resulting cast is cleaned of possible tissue/
reagent debris and examined in SEM. Apart from revealing the topographical pattern of
microvasculature, the casts can also provide information concerning luminal differentia-
tions, such as bulging endothelial cell nuclei, fenestrated capillary areas, and valves or
local constrictions reflecting the presence of contracted smooth muscle cells or pericyte
processes, since the cast surface replicates in detail the luminal surface of blood vessels.
In theory, corrosion casting is suitable to replicate all luminal systems or spaces of the
organism, but its applications to studies of organs/systems other than blood or lymphatic
vessels encounter significant technical difficulties and have been very rare.

2.2 History

Although corrosion casting is now regarded as one of the material preparation techniques
for SEM, it has its roots in gross anatomy. Invention of a casting substance, capable of
filling major body spaces and blood vessels and subsequent hardening in situ, resistant
to mechanical and chemical damage and thus appropriate to create durable specimens
that can be presented to students, had been an anatomist’s dream for many years. The idea
of using corrosion casts for gross anatomical studies is a few hundred years old. Leonardo
da Vinci (1452–1519) made wax casts of brain ventricles and heart chambers of humans.
Jan Swammerdam (1637–1680) is commonly mentioned as the inventor of solidifying
injection mass (although he also used melted wax) and syringe to perform injections of
blood vessels. At the turn of the seventeenth and eighteenth century and later, different
casting media were tried: Gottfried Bidloo in 1685 used melted metal to inject trachea

Scanning Electron Microscopy for the Life Sciences, edited by H. Schatten. Published by Cambridge
University Press © Cambridge University Press 2012
 Corrosion casting technique 17

Figure 2.1 Microvasculature of poison gland in the skin of salamander. Reprinted with kind permission from
Springer Science+Business Media, from: Miodoński, A. and Jasiński, A. (1979) Scanning electron
microscopy of microcorrosion casts of the vascular bed in the skin of the spotted salamander,
Salamandra salamandra L. Cell Tissue Res., 196, 153–62.

and bronchi, 70 years later Johannes N. Lieberkühn employed a mixture of 1/10 natural
resin and 9/10 turpentine and was the first to produce successfully injected microvessels
of the gastrointestinal tract mucosa.
The corrosion casting era was opened by Ruysch (1725) and Lieberkühn (1748) who
injected human organs with casting medium and then used insect larvae to corrode the
injected specimens. By the end of the nineteenth century Hyrtl, Schiefferdecker,
Teichmann, Hoyer Sr. and Jr., Gerlach, Voigt, Storch, and Kadyi continued the systemic
anatomical studies using different kinds of casting media such as gelatin, celloidin,
cellulose, or modified glazier’s putty (Kuś, 1969). The latter medium was employed by
Teichmann, one of the most outstanding pioneers of casting techniques, famous for his
unique cast specimens presently exhibited in the museum of Chair of Anatomy,
Jagiellonian University, Krakow, Poland.
The currently used casting media – synthetic resins – had their precursors in the first
half of the twentieth century. In 1935, Schummer introduced a polymerizing resin called
Plastoid and injected testis and ureter (Schummer, 1935). In 1936, Narat et al. used for
the first time vinyl-polychloride for injection of placental, renal, and splenic blood
vessels. Significant technical progress was achieved in the 1950s and later, when
synthetic resins were introduced as casting media: acrylic resin (Taniguchi et al. in
1952); polyester resin (Aleksandrowicz and Łoziński in 1959); vinyl chloride (Goetzen
in 1966); mixture of methylmethacrylates; methylmethacrylate (Murakami in 1971);
Batson No. 17 (Nopanitaya et al. in 1979); Araldite CY 223; Tardoplast (Amselgruber
and König in 1987), (Lametschwandtner et al., 1990).
Another type of casting medium tested in the 1970s was latex and silicone rubber
(Cementex, Microfil). However, rubber casts showed extreme fragility and easily dis-
integrated during corrosion. They required freeze-drying or critical point drying to
maintain their three-dimensional arrangement and did not replicate luminal surface
microstructures consistently.
18 Jerzy Walocha, Jan A. Litwin, and Adam J. Miodoński

Recently, a new polyurethane-based PU4ii resin has been proposed as an optimal

casting medium (Krucker et al., 2006).
The corrosion media used to produce the casts included various alkalis (NaOH, KOH,
sodium hypochloride) and acids (HCl, H2SO4, HNO3, bichromate sulfuric acid, chro-
mium trioxide, and formic acid), although it has been shown that some of them can cause
severe damage to the casts and that different casting substances require different corro-
sion media to prevent such damage (Hodde et al., 1990).
Forty years ago, Janice Nowell and her coworkers for the first time used a combination
of corrosion casting and observation of the casts in SEM to study the structure of avian
lungs (Nowell et al., 1970). One year later, Murakami (1971) applied that approach to the
study of vascular networks. Since then, over 2000 papers presenting scanning electron
microscopy of corrosion casts in animals, humans, and even invertebrates have been
published. The data provided by this technique were helpful not only in evaluating
microanatomy of the studied systems, but also in elucidating some aspects of develop-
ment, differentiation, aging, and pathological processes.

2.3 Corrosion casting procedure

The corrosion casting/SEM technique consists of several stages:

* washing of the space to be cast in order to remove its content (e.g. blood)
* injection of casting medium
* hardening of casting medium
* corrosion (maceration)
* dissection (optional)
* cleaning and drying
* mounting and conductive treatment
* SEM examination
* quantitative analysis of SEM images (optional).
Since the vast majority of corrosion casting/SEM studies has been performed on blood
vessels, the technical aspects are discussed in this context.
In small animals, a cast can be made of the entire vascular bed. Larger animals require
casting of selected vascular systems, usually those of organs chosen for the study, by
injecting the casting medium via their main supplying vessels and allowing outflow of
the medium via the main draining vessels. This can be done either in situ or after removal
of the organ. It is often difficult to obtain such casts of an acceptable quality. Larger
volumes of rinsing and casting media have to be used, there is a risk of vasoconstriction
and intravascular coagulation before the casting step leading to incomplete replication,
and furthermore, casts can be easily damaged during specimen handling, especially when
cast dissection is necessary. The former risk can be minimized if organs are perfused
in situ in an anesthetized animal and if the perfusion solution contains anticoagulants.
Biomedical research on humans is particularly demanding, since casting has to be carried
out in material obtained post-operatively or on autopsies. Fresh post-operative specimens
 Corrosion casting technique 19

of human organs can only rarely be used because whole organs are required for resin
injection, while at least fragments of the organ must be collected for histopathological
examination. Corrosion casting of autopsy material bears a risk of poor tissue preserva-
tion and endothelial cell necrosis. For obvious reasons, the completeness of replication
and number of artifacts depend on the time elapsing between the interruption of the
circulation and filling of vessels with the casting medium. Several authors have reported
successful post-mortem casting and SEM analysis of human microvascular systems
(Karaganov et al., 1981; Banya et al., 1989; Murakami et al., 1992; Walocha et al.,
2003; 2012). They were able to obtain acceptable casts from organs collected upon
autopsy within 24 hours after death. Acceptable post-mortem vascular casts have also
been obtained in large animals (Martin-Orti et al., 1999).
There are two main obstacles that can hamper the complete filling of the vascular
system with casting medium: blood clots and vasoconstriction caused by metabolic or
neurogenic factors. Therefore, the first step of the procedure – perfusion of the vessels
with a washing solution is crucial for optimal replication. The solution (PBS, Tyrode’s
solution, Ringer’s solution) should contain anticoagulant (heparin) and a spasmolytic
agent (lidocaine, papaverine). Subsequent perfusion with a low-concentration fixative,
e.g. formaldehyde or glutaraldehyde (vascular fixation) is optional, but fixation of the
vascular walls is believed to increase their stability and to prevent ruptures leading to
extravasation of the casting medium or dilatation of the vessels under its pressure.
The properties of the casting medium are another key factor for high-quality casts. An
ideal casting medium:
* has relatively low viscosity (as close to that of body fluids as possible) to ensure perfect
filling of the smallest spaces
* is chemically and physiologically neutral in the system to be cast
* polymerizes within an appropriately short time (3–15 minutes)
* does not shrink/deform during polymerization and drying
* allows microdissection of the cast without breaking or deformation
* is fully resistant to corroding reagents (cast shows no surface damage)
* resists electron bombardment during SEM examination
* allows replication of minute details of cast surfaces
* reveals no toxicity.
The casting media currently in use are not perfect – some of the above listed criteria are
only partially fulfilled (e.g. there seems to be an inverse correlation between viscosity of
the liquid resin and its shrinkage rate during polymerization) – but still they provide
satisfactory replication at magnifications offered by SEM. The most widely employed
media are methylmethacrylates: Mercox, Batson’s no. 17, and Technovit 8001. The
casting kit usually includes the resin, polymerization catalyst (plasticizer), and some-
times polymerization promoter (initiator/accelerator).
Perfusion of specimens with casting medium should be performed under controlled
pressure, since too low a pressure usually leads to incomplete replication, while too high
a pressure, albeit minimizing that hazard, can induce deformation and damage of the
vascular walls resulting in casting artifacts such as bulges and extravasations. Manual
20 Jerzy Walocha, Jan A. Litwin, and Adam J. Miodoński

perfusion is the simplest, but it cannot be standardized and most authors use specially
designed mechanical injection/perfusion devices, which allow injection of preprog-
rammed volumes of casting medium per minute at controlled injection pressure.
Polymerization of the resin is possible at room temperature, but it is usually carried out
at elevated temperature by placing the specimen in a water bath at 40 to 60 °C for a few
hours. Such treatment accelerates polymerization and allows obtaining less fragile casts.
Corrosion of tissues surrounding the cast is mostly performed in solutions of sodium or
potassium hydroxide at room temperature or at 37–39 °C. Although a wide range of
hydroxide concentrations (15–60%) has been used in corrosion casting studies, it seems
that the optimal concentration is 5–20%, since concentrations of 40% and more can
inhibit the maceration process by saponification of proteins (Hodde et al., 1990).
Corrosion is a time-consuming process and in larger specimens can take a few days. It
can be accelerated by relatively frequent changes of the hydroxide followed by washes
with gently running warm tap water.
Small casts can be directly cleaned and mounted for observation in SEM. In most
cases, however, the size of cast obtained from an injected organ requires its dissection,
not only to cope with the space available in the SEM specimen chamber, but also to reveal
deeper regions of the cast. Dissection should not alter natural shape and three-
dimensional topography of the cast. It can be carried out before or after corrosion.
Dissection performed before corrosion has an advantage of the cast being supported by
the surrounding tissues with distinguishable anatomical landmarks. Dissection after
corrosion requires particular precision and delicacy – it can be performed with the use
of a microtome blade, microsurgical tools or by low-power laser beam, although the latter
can produce thermal artifacts at the dissection plane. When highly precise dissection of
small areas is needed, it can be done under a stereomicroscope on a dried and mounted
cast, in a warm alcohol bath, or even using a micromanipulator associated with SEM
(Lametschwandtner et al., 1990). However, the risk of cast damage is lower when prior to
dissection the cast is stabilized by embedding in a solidifying medium, such as water-
soluble wax (e.g. Aquax – Miodoński et al., 1980) or a mixture of polyethylene glycols
(Walocha et al., 2002), which do not interfere chemically with the casting medium.
After dissection, the surface of the cast should be cleaned to remove possible tissue/
reagent debris. This can be achieved by immersing the cast in 5–10% trichloroacetic acid,
2% HCl or 2–3% formic acid, although other cleaning media, such as HCl-collagenase
solution or alcohol, have also been used. This step is completed by a wash in tap water
followed by distilled water, which removes all salts that might later crystallize on the
drying replica.
Casts can be dried in air, although tension exerted on the cast during evaporation of
water increases the risk of local deformations (Lametschwandtner et al., 1990); hence a
short rinse in ethanol is recommended to minimize that effect. Freeze-drying or, less
commonly used, critical point drying are useful options.
The casting media are not electron conductive, hence the casts have to be mounted on a
metallic support by a conductive mediator and then metal-coated. Silver tape or adhesive
tape coated with silver colloid have been used for mounting, although in the latter case
the colloid can be adsorbed by adjacent regions of the cast, hampering SEM examination.
 Corrosion casting technique 21

“Conductive bridges” introduced by Lametschwandtner et al. (1980) reduce specimen

charging during SEM examination and allow a relatively safe remounting of the cast.
Such bridges are usually made of fine copper wires attached with silver glue to various
regions of the cast on one end and to the support on the other. Their number, depending
on the size of the cast, ranges from one for small casts (using a modified support and
relatively thick wire, the cast can be rotated to allow SEM examination of its “bottom”) to
about twenty for very large casts. Recently, Verli et al. (2007) used carbon tape to connect
the wires to the mounting platform.
The casts are usually coated with gold, often with an underlying layer of carbon,
although other metals, such as chromium, platinum, palladium, and osmium have also
been used (Belz and Auchterlonie, 1995). Coating can be performed by sputtering or
evaporation. Sputtering seems to be safer, since the heat associated with evaporation can
damage the cast. If the cast is large, coating should be repeated several times, to allow
penetration of the metal to the deepest regions of the specimen. With time, fissures and
cracks can appear in the coating layer, so the casts should be examined in the SEM
relatively shortly after coating. After longer storage, casts may require recoating.

2.4 Microvascular casts – interpretation of SEM images

The quasi-3-D appearance of cast microvascular systems in SEM facilitates their analysis
based on continuity of successive vessel types (arteries – arterioles – capillaries –
venules – veins). However, casts do not allow the investigator to identify direction of
blood flow, thus discrimination between arteries/arterioles and veins/venules is crucial
for interpretation of the vascular bed (Figure 2.2). It was Miodoński et al. (1976) who
first noticed that nuclear imprints of arteries were different from those of veins. The
surfaces of arterial casts exhibit ovoid or fusiform nuclear imprints, with their long
axes oriented along the long axis of the vessel. The imprints, quite regularly distributed,
appear as sharply demarcated depressions. In the veins, imprints are roundish, shallower,
less sharply outlined, and less regularly distributed on the cast surface (Figure 2.3). In
capillary casts, the nuclear imprints are less distinct or even absent (the surface can look
smooth). The shape of endothelial cells can also be observed, since the intercellular
borders are visible as delicate furrows. In the arteries, endothelial cells are elongated
and rhomboidal, oriented according to the long axis of the vessel, whereas in veins
the cells are more polygonal. Later studies have confirmed these observations (Gnepp
and Green, 1979; Nopanitaya et al., 1979). The branching pattern can also be helpful
in distinguishing arterioles from venules: an arteriolar branching pattern is mostly
symmetrical, with branches showing a similar diameter, whereas a venular branching
pattern is in many cases asymmetrical, with the venules receiving tributaries nearly as
large as the draining venule or as small as capillaries (Hodde et al., 1977; Christofferson
and Nilson, 1988).
Apart from discrimination between arteries/arterioles and veins/venules, the identifica-
tion of different microvessel types according to classification based on their wall structure –
arterioles, terminal arterioles (metaarterioles), capillaries, postcapillary venules, collecting
22 Jerzy Walocha, Jan A. Litwin, and Adam J. Miodoński

Figure 2.2 An example of a closed microvascular system. Differentiation between artery (A) and vein (V) is
necessary to determine direction of blood flow. Reprinted with kind permission from Springer
Science+Business Media, from: Miodoński, A.J. and Bär, T. (1987) The superficial vascular
hyaloid system in eye of frogs, Rana temporaria and Rana esculenta. Scanning electron-
microscopic study of vascular corrosion casts. Cell Tissue Res., 250, 465–473.

Figure 2.3 Endothelial nuclear imprints in cast of artery (A) and vein (V). Note differences in their shape,
appearance, and distribution. Reprinted with permission from Miodoński, A., Kuś, J., and
Tyrankiewicz, R. SEM blood vessel cast analysis. In: Three-dimensional Microanatomy of Cells
and Tissue Surfaces (ed. Allen, D.J., DiDio, L.J.A., and Motta, P.M.), Amsterdam, Elsevier, 1981,
pp. 71–87.

venules, muscular venules, and small collecting veins – is practically limited in the casts to
estimation of the vessel diameter and should be treated with caution.
The casts also visualize details of luminal topography of blood vessels reflecting some
structures associated with the vascular wall. Venous valves appear in the casts as slight
 Corrosion casting technique 23

Figure 2.4 Arteriole with a sphincter (S) at the site of its origin and with shallower circular imprints of smooth
muscle cells (white arrows). In a nearby capillary, two narrow imprints of pericyte processes can be
seen (black arrowheads).

expansions at valve sinuses and deep slits at the sites of valve leaflets. Valvular malfor-
mations, e.g. leaflets shared by adjacent valves, can also be easily observed (Hossler and
West, 1988).
Continuous capillaries can be distinguished from fenestrated ones: casts of the former
are straight and uniform in diameter, whereas casts of the latter are undulating and show
eccentric smooth-surfaced dilatations alternating with narrower segments. Although
fenestrations themselves are too small to be replicated, comparative SEM and TEM
analysis has revealed that the bulging cast segments corresponded to the fenestrated areas
of capillaries (Aharinejad and Böck, 1994).
Thin, annular or semilunar grooves observed on the cast surfaces of capillaries and
postcapillary venules reflect local constrictions of the capillary/postcapillary wall
induced by pericyte processes located on the outer surface of the endothelial lining
(Aharinejad and Böck, 1992). Accordingly, wider annular constrictions observed in
arteriolar or venular casts correspond to contracted smooth muscle cells, and if the
constriction is deep, it suggests the presence of a sphincter (Aharinejad et al., 1992) or
intra-arterial cushion (Matsuura and Yamamoto, 1988) (Figure 2.4).
Scanning electron microscopy of corrosion casts can also reveal some structural
features of the capillary bed indicative of angiogenesis. Short, blind “capillary sprouts”
have been commonly interpreted as marks of ongoing angiogenesis, although incomplete
replication should also be taken under consideration in such cases. Small, smooth “holes”
ranging from 0.5 to 2 µm in diameter (Figure 2.5) are characteristic of another angiogenic
process, the intussusceptive capillary growth (Zagórska-Świeży et al., 2008), character-
ized by formation of transcapillary tissue pillars (Burri et al., 2004).

2.5 Quantitative analysis of vascular corrosion casts in SEM

An early and simple method of quantification was weighing the cast of a vascular system
fully replicated with resin of a known density – it permitted calculation of the vascular
bed volume (Weiger et al., 1986).
24 Jerzy Walocha, Jan A. Litwin, and Adam J. Miodoński

Figure 2.5 Capillary network with features of angiogenesis: blind capillary sprouts (white arrows) and holes
indicative of intussusceptive capillary growth (black arrowheads).

Morphometry, in the last decades assisted by computerized image analysis, enables

measurements of various vascular system parameters such as interbranch and inter-
vascular distances, branching angles, vessel lengths and diameters – combination of
these parameters quantitatively defines three-dimensional (3-D) structure of the vas-
cular bed.
For a long time the measurements had been carried out on two-dimensional SEM
micrographs and translation of results into 3-D was problematic. Minnich et al. (1999)
presented a method for accurate dimensional and angular measurements of miscrostruc-
tures inspected in SEM. Their approach included collection of stereopaired digital
images from SEM and creation of 3-D image representations, as well as setting of
measuring points in the images, computation of precise space coordinates from the
corresponding point coordinates, vector equation determination of distances and angles
between the consecutive corresponding points, and processing of data by analysis
software.
This method was further developed to quantify bifurcation indices, area ratios, and
asymmetry ratios, and to test the bifurcations for the optimality principles. The obtained
data allowed drawing some functional conclusions concerning bifurcation pattern in the
studied vascular system, which favored minimum pumping power and minimum volume
rather than minimum surface and minimum drag (Lametschwandtner et al., 2005).
Manelli et al. (2007) described an automatic 3-D reconstruction of vascular casts directly
from stereo-images and developed software that performed morphometric measurements
on such 3-D constructs.
Recently, “true” 3-D visualization and quantification of corrosion cast microvascular
networks was achieved by using microcomputed tomography (micro-CT) instead of
SEM images. The resolution of micro-CT images is inferior to that of SEM, but fully
sufficient for studying the spatial distribution of microvessels. This approach made
possible a highly precise reconstruction of the microvascular topography and its multi-
parameter analysis (Heinzer et al., 2006; Atwood et al., 2010).
 Corrosion casting technique 25

2.6 Corrosion casting studies of microvascular systems

Studies of vascular systems using corrosion casting and SEM have included three major
fields: architecture of normal vascular systems, architecture of developing vascular
systems, and vascular systems in experimental and clinical pathology.
An impressive – nearly complete – list of papers published in the first two decades of
vascular corrosion casting/SEM research was presented in the fundamental review by
Lametschwandtner et al. (1990). Since then, hundreds of studies using that technique
have appeared in the literature – the interested reader can access them via the Pubmed
website.
Normal mature and developing vascular systems have been investigated in a wide
spectrum of animal species and in humans. Human developmental studies are relatively
rare because of difficulties in collection and processing of fetal material, although
Miodoński’s group published a series of papers presenting developing vasculature of
various organs in the second trimester of fetal life (e.g. Zagórska-Świeży et al., 2008).
Corrosion casting has also been employed occasionally to study lymphatic vessels and
lymphatic spaces (Castenholz and Castenholz, 1996; Okada et al., 2002; Schraufnagel
et al., 2003).
In biomedical research, SEM studies of vascular corrosion casts have included such
topics as reconstructive medicine and vascular changes in a variety of pathological
processes, with special emphasis on formation and remodeling of microvascular net-
works in tumors (Skinner et al., 1995).
The technique has provided valuable information concerning vascularization of var-
ious types of flaps used in plastic/reconstructive surgery (skin flaps, musculocutaneous
perforator flaps, mucoperiosteal flaps, venous flaps) (Bergeron et al., 2006).
Dilatation of capillary plexuses was observed in inflammation (Ravnic et al., 2007). In
cerebral ischemia, casting revealed avascular areas, arteriolar vasospasm, interrupted
arteriolar branches and thin, “stringy” capillaries (Ohtake et al., 2004). Choroidal and
retinal vasculature of hypertensive rats showed tortuosity, irregularity, and narrowing of
arteries and capillaries (Bhutto and Amemiya, 2002). In cholesterolemia, alterations
observed in retinal vessels included straight, string-like capillaries as well as elongation
and straightening of precapillary arterioles (Yamakawa et al., 2001). Numerous anoma-
lies of retinal vessels were found in diabetes: arteries were tortuous, narrow, and irregular
in diameter, precapillary arterioles formed hairpin loops, venules were sparse and
capillaries showed undulations, loops, and general narrowing with local dilatations
described as microaneurysms (Bhutto et al., 2002). Loss of typical capillary pattern
and decrease in the number of feeding arterioles in the optic nerve, as well avascular areas
in choroid and retina were demonstrated in glaucomatous eyes (Zhao and Cioffi, 2000).
In cirrhotic liver, the distance between pre- and post-sinusoidal vessels was reduced and
newly formed vessels appeared in the hepatic tissue, some of them connected pre- and
post-sinusoidal vessels bypassing the sinusoids, others formed perinodular plexuses
(Gaudio et al., 1993). Disarranged microvascular patterns with features of regression
and formation of dense plexuses around the cysts were observed in polycystic kidney
26 Jerzy Walocha, Jan A. Litwin, and Adam J. Miodoński

disease (Wei et al., 2006). In a mouse model of Alzheimer’s disease, brain microvessels
showed alterations even before the formation of amyloid plaques and at more advanced
stages of the disease areas occupied by plaques were avascular (Meyer et al., 2008).
Differentiated microvascular patterns were revealed by corrosion casting and SEM in
tumors, both transplanted into animals and surgically removed from patients or collected
at autopsy. In the former, a relatively simple angioarchitecture was usually observed, with
numerous angiogenic capillary sprouts and variable capillary diameter as well as inter-
vessel and interbranch diameters (Grunt et al., 1986; Konerding et al., 1999; Tsunenari
et al., 2002; Sangiorgi et al., 2006). Human tumors (hepatocellular carcinoma, malignant
melanoma, colorectal cancer, bladder cancer, renal clear cell carcinoma, larynx cancer,
leiomyomas) injected with casting media after resection showed more complex vascular
architecture, which was rather specific for the tumor type (Konerding et al., 2001), but
shared such common features as increased and/or irregular capillary diameter and signs
of active angiogenesis. Some tumors revealed an outer vascular “coat” or “capsule” with
very high vessel density and less vascularized central areas (Grunt et al., 1986; Bugajski
et al., 1989; Walocha et al., 2003). Unusual capillary patterns – flattened “sheets”,
multiple, sometimes tortuous loops, glomerular or basket-like arrays (Figure 2.6) –
were frequently observed in various tumor types (Miodoński et al., 1980, 1998;
Arashiro et al., 1995; Skinner et al., 1995; Arashiro, 2002). Hepatocellular carcinoma
nodules were supplied by peripheral vascular plexuses containing branches of both
hepatic artery and portal vein and their intrinsic vessels communicated with sinusoids
of the surrounding normal hepatic tissue. This multiple blood supply explains survival of
cancer cells after arterial embolization (Kita et al., 1991).
In spite of morphological differentiation, quantitative comparison of vascular network
parameters in 13 experimental and 3 human tumors of different origin showed a high
degree of similarity (Konerding et al., 1995).

2.7 Other applications of corrosion casting/SEM to life sciences

In contrast to innumerable papers devoted to vascular corrosion casts, surprisingly few

authors have tried to apply this technique to other systems or organs. The scarcity of such
studies has probably resulted from considerable difficulties in injecting “blind” tubular
systems such as ducts of glands or respiratory passages. However, some attempts have
been successful. Replication of air passages allowed study of the three-dimensional
topography of animal airways (Nowell et al., 1970; Nettum, 1996) as well as human
respiratory acinus and its development during the fetal period (Dilly, 1984). Corrosion
casting combined with SEM revealed the spatial arrangement and microanatomical
details of the biliary tree (Gaudio et al., 1988), pancreatic ducts (Ashizawa et al.,
1997), and duct system of mammary gland (Schenkman et al., 1985). In the eye, drainage
routes for aqueous humor have been replicated (Ujiie and Bill, 1984; Krohn and
Bertelsen, 1997). Corrosion casting of a brain ventricle system allowed visualization of
not only its spatial topography, but also local variations in the ependymal lining (Liao and
Lu, 1993). Injection of kidney with a resin via a ureter produced casts of collecting
 Corrosion casting technique 27

Figure 2.6 Extremely dense, sheet-like and glomerular capillary plexuses in exophytic portions of urinary
bladder cancer. Reprinted with kind permission from Springer Science+Business Media, from:
Miodoński et al. (1998).

tubules and Henle’s loops of sufficient quality to study their spatial arrangement and
dimensions (Magaudda et al., 1990). In the reproductive system, corrosion casting has
revealed the successive segments of the efferent ductules (Stoffel et al., 1991) and
changes in the patency of the utero-oviductal tract in the course of an estrous cycle
(Doboszyńska and Sobotka, 2002). Okada et al. (2002) studied macerated compact bone
and succeeded in obtaining corrosion casts of not only vascular canals but also minute
structures such as bone lacunae and canaliculi, demonstrating age-associated changes in
their shape and distribution. Even more impressive results were presented by Meyer
(1989), who replicated the insect tracheal system and was able to visualize even the
thinnest tracheoles, approx. 70 nm in diameter.

2.8 Concluding remarks

Corrosion casting combined with SEM is now a well-established method used in many
areas of life science research, allowing three-dimensional observation of large-scale
28 Jerzy Walocha, Jan A. Litwin, and Adam J. Miodoński

specimens. Although technically demanding, it does not require sophisticated equipment

and can be carried out in any SEM laboratory. Future perspectives seem to be focused on
introducing new low-viscosity casting media, which would precisely replicate very small
structures present on the luminal surfaces of the cast spaces, such as gold particles used as
labels of antibodies or lectins. With the help of high-resolution SEM, this could make
possible combination of corrosion casting with immunocytochemistry or lectin histo-
chemistry and broaden the scope of corrosion casting research to studies on distribution
of specific molecules (e.g. receptors) on the cast surfaces.

2.9 Acknowledgments

The authors thank J. Urbaniak and K. Zagórska-Świeży for skillful technical assistance.

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30 Jerzy Walocha, Jan A. Litwin, and Adam J. Miodoński

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3 Revealing the internal structure of
cells in three dimensions with
scanning electron microscopy
Sol Sepsenwol

3.1 Introduction

Scanning electron microscopy (SEM) is ideally suited to imaging structures with complex
topology. The limitation of SEM is that it can only give information about the surface layer
of the sample. In biology, many structures of interest are internal, so that the problem is to
open up the cell or extracellular structures, preferably without disturbing their normal
relationships in the intact cell. With the advent of high-resolution, low-voltage, field
emission scanning electron microscopy (HRSEM) and special preparation techniques, it
is possible to view both internal and external macromolecular structures in three dimen-
sions, thereby restoring depth information lacking in conventional microscopic studies.
HRSEM has important advantages over transmission electron microscopy (TEM) methods
used to visualize three-dimensional (3-D) structures at high resolution. TEM is limited by
sample thickness (≤ 1.0 micron), even at high accelerating voltages, and by beam damage
to delicate structures, such as cytoskeletal fibers, especially when unsupported by plastic
embedment. The deep etch freeze–fracture metal replica techniques for TEM (Heuser,
1976; Henser and Salpeter, 1979) work beautifully to circumvent some of these thickness
limitations1 but require, among other things, a cryogenic evaporative metal coating system
not available in most EM labs. Cryo-TEM tomography likewise can create detailed views
of cells to a depth of a few hundred nm. HRSEM is not so limited and does not require the
computational reconstruction of tomography. Newer HRSEM can produce nanometer
scale resolution, 3-D images at accelerating voltages below 1 kV (Ris, 1991; Lück et al.,
2010). The relatively simple methods described here for direct visualization and immuno-
labeling of the internal structures of cells or extracellular structures at high resolution in
three dimensions should be generally useful in exploring complex internal cell architec-
tures. Most of the techniques below were developed to study intracellular ultrastructure of
cells either in monolayers on substrate or in suspension, but can be readily adapted to tissue

1
See the Heuser Lab website (http://www.heuserlab.wustl.edu) for a dazzling gallery of deep-etch TEM
images, many unpublished.
Note: seven of the images in this chapter are presented in two-color anaglyph stereo in the color plate section
and should be viewed with red–green decoding glasses (red – left eye). Two-color glasses are readily available
from EM supply houses and stereo specialty houses.

Scanning Electron Microscopy for the Life Sciences, edited by H. Schatten. Published by Cambridge
University Press © Cambridge University Press 2012
34 Sol Sepsenwol

samples. These techniques are not restricted to HRSEM, but may also be used with lower
resolution, tungsten filament SEMs. They are: (a) detergent extraction, (b) cleaving on
scored glass, (c) tape ripping, (d) agar/alginate string fracturing, (e) polylysine wet ripping
and immunolabeling, (f) de-embedding of semi-thick plastic sections, (g) sectioned fresh
material on a freezing stage, and (h) high-pressure freezing and cryo-SEM.

3.2 Notes on fixation and dehydration of samples

3.2.1 Fixation
Fixation, of course, is critical to reliable preservation of cell ultrastructure and also affects
immunolabeling. In the writer’s lab, samples are usually fixed in freshly made 1–2% EM
grade glutaraldehyde in compatible salt solution containing 50 mM HEPES buffered
between pH 6.8 and 7.8. For most mammalian cells, this can be based on a pH 7.2–7.4
HEPES saline buffer with added calcium, magnesium, and other salts. Sometimes
0.025% saponin is added to the fixative to permeabilize cells for more rapid fixation of
internal structures. Cells are usually post-fixed with freshly made 0.1% osmium tetroxide
in the same buffer to provide rigidity to the structures, as well as an enhanced secondary
electron signal. The light osmium post-fixation will not obscure back-scatter detection of
colloidal gold immunolabeling, given the newer high-sensitivity BSE detectors. For
structures known to be labile during fixation at 0–25 °C, flash freezing and freeze
substitution osmium fixation can be used (Buser and Walther, 2008).

3.2.2 Dehydration
Meticulous dehydration is as essential to reliable preservation of internal structures as
fixation, since vanishingly small amounts of water can cause adherence between adjacent
protein structures, leading to artifacts that may be beautiful, but biologically irrelevant
(Small, 1981). Good dehydration can be achieved by attention to the following: (a)
ethanol dehydration times should be longer than 10 min per change with continuous,
gentle agitation, (b) de-fined alumina dehydrating agents (molecular sieve) should be
used in the absolute ethanol stock bottle, (c) exchanges of liquid CO2 during critical point
drying (CPD) should be extensive with agitation, (d) a molecular sieve trap should be
used in the liquid CO2 line, (e) chambers or racks should allow free flow of liquid CO2
over the sample (Sepsenwol, 2005). In the writer’s experience, other solvent dehydration
systems such as hexamethyldisilazine (HMDS) or Pel-Dri® do not produce results
comparable to CPD for high-resolution work.

3.3 Detergent treatment to expose internal cell structure demembranation

during fixation

This technique has been used to investigate internal cytoskeletal architecture in

cells attached to substrate by treating the cells with a nonionic detergent (usually
 Revealing the internal structure of cells 35

Triton X-100) in a buffer known to stabilize actin and intermediate filaments (Schliwa
and van Blerkom, 1981). While detergent treatment has its own peculiar artifacts, one is
desirable – the removal of the overlying plasma membrane and most of the cytoplasmic
organelles and unstable polymers, which can obscure the overall superstructure of the
assembled cytoskeleton and organelles associated with the attached surface of the cell.
In so doing, it creates a simplified view of the cytoskeleton, which may be useful for
understanding the much more complex, dynamic cytoskeleton in situ. The author uses
fixative and detergent together, 1% glutaraldehyde containing 0.1% Triton X-100
detergent in a HEPES isotonic saline buffer, followed by buffer rinses and 0.1%
osmium post-fixation in the same buffer. Because the amount of disruption of the
detergent is sensitive to shear forces, time, and fixative, it is hard to create consistent
results, even within a single sample. Detergent fixation with high voltage, 1000 kV,
transmission EM (HVEM) was originally used to demonstrate a system of fiber
complexes in crawling sperm of the nematode, Ascaris suum (Sepsenwol et al.,
1989). Figure 3.1 (and color plate) compares the results of detergent treatment as
imaged by HVEM and by HRSEM. The HVEM image (Figure 3.1A) shows the

Figure 3.1 Comparison of high-voltage TEM (HVEM) with high-resolution SEM of triton-treated, fixed,
crawling sperm of the nematode intestinal parasite, Ascaris suum. (A) Activated sperm cell
crawling on coated grid, fixed and CPD. The pseudopod has been de-membranated, revealing the
“bottlebrush” arrangement of major sperm protein filaments into fiber complexes (fc) that represent
its machine for crawling. Because of thickness limitations of TEM, most of the cytoskeleton was
removed, so that the full array of complexes cannot be seen. Accelerating voltage (Vacc)
= 1000 kV; in stereo, left eye red. (B) This cell is from the same HVEM preparation, but Pt-coated
and viewed with HRSEM. The branching and composition of the fiber complexes are clearer, as
is the loss of all but the adherent branches of the cytoskeleton caused by the detergent treatment.
It also demonstrates the integrity of the cell–body plasma membrane, which is resistant to detergent
disruption, and some of its unique surface features. Vacc = 1.5 kV; in stereo, left eye red. (See color
plate section for two-color stereo 3-D version.)
36 Sol Sepsenwol

basic fiber complex arrangement: complexes branching from their origins at the leading
edge of the pseudopod. Most of the features of the cell body, about 5 μm thick, are not
visible with HVEM. The HRSEM of the same preparation (Figure 3.1B) shows not
only the loss of pseudopod components, including parts of the cytoskeleton itself, but
also details of the cell body and its unique membrane. While detergent extraction may
be useful for simplifying the basic structures left behind, inevitably other methods are
necessary that better preserve in vivo architecture and its special relationships to other
parts of the cell.

3.4 Cleaved glass fracturing

This simple method provides a side view of attached cells and is useful for studying
cell-to-substrate or cell-to-cell contacts. Occasionally, it can provide sections through
cells to show internal components associated with attachment. Cells are cultured on
finely scored glass, the glass is then broken along the scores and mounted edge on for
SEM observation. Specifically, acid cleaned #2 thickness coverslips are lightly scored
with a diamond scribe into 1 mm lanes, sterilized, placed in a culture dish, overlaid
with cell suspension, and cultured. Slips with attached cells are glutaraldehyde- and
osmium-fixed, CPD, then cleaved into 1 mm strips with fine forceps. The strips are
mounted on edge to double-sided conductive tape on a conductive base and sputter
coated with Pt (see inset to Figure 3.2). This produces useful side views of the cells
and their special types of contact with the substrates. When cells attach across score
lines, cleaving creates a section through the cell that allows a detailed view of

Figure 3.2 Cleaving attached cells on glass. Ascaris sperm crawling on scored glass are fixed, CPD, Pt-coated,
and mounted edge on. Crawling Ascaris sperm, shown in side view 15 min after activation, is
fully attached to glass. The individual points of attachment, the villipodia, spread out along the
bottom to form a continuous sheet of contact. This cell has been cleaved along the glass score,
showing that the fibrous cytoskeleton of the pseudopod underlies the cell body so that the cell body
rides above the pseudopod, rather than behind it. The cell in the background is forming a
pseudopod and has not yet attached to the glass. Vacc = 1.5 kV. (Inset) A 1 mm coverslip strip
mounted on edge on a silicon chip with conductive tape.
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The Project Gutenberg eBook of The three taps
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Title: The three taps

A detective story without a moral

Author: Ronald Arbuthnott Knox

Release date: March 18, 2024 [eBook #73198]

Language: English

Original publication: New York, NY: Jacobsen Publishing Company,

Inc, 1927

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*** START OF THE PROJECT GUTENBERG EBOOK THE THREE TAPS

***
 THE THREE TAPS

A Detective Story without a Moral

by

RONALD A. KNOX
 Contents
I The Euthanasia Policy
II The Detective Malgré Lui
III At the Load of Mischief
IV The Bedroom
V Supper, and Mr. Brinkman
VI An Ear at the Keyhole
VII From Leyland’s Note-Book
VIII The Bishop at Home
IX The Late Rector of Hipley
X The Bet Doubled
XI The Generalship of Angela
XII The Makings of a Trap
XIII A Morning with the Haberdasher
XIV Bredon Is Taken for a Walk
XV A Scrap of Paper
XVI A Visitor from Pullford
XVII Mysterious Behaviour of the Old Gentleman
XVIII The Barmaid Is Brought to Book
XIX How Leyland Spent the Evening
XX How Bredon Spent the Evening
XXI How Eames Spent the Evening
XXII At a Standstill
XXIII Leyland’s Account of It All
XXIV Mottram’s Account of It All
XXV Bredon’s Account of It All
 DEDICATED TO

SUSAN AND FRANCIS BAKER

(only he mustn’t sit up too late over it)
 Chapter I.
The Euthanasia Policy
The principles of insurance, they tell us, were not hidden from
our Anglo-Saxon forefathers. How anybody had the enterprise in
those rough-and-tumble days to guarantee a client against “fire,
water, robbery or other calamity” remains a problem for the
historian; the more so as it appears that mathematical calculations
were first applied to the business by the eminent John de Witt. In
our own time, at any rate, the insurance companies have woven a
golden net under the tight-rope walk of existence; if life is a lottery,
the prudent citizen faces it with the consciousness that he is backed
both ways. Had the idea been thoroughly grasped in those remoter
periods, no doubt but Alfred’s hostess would have been easily
consoled for the damage done to her cakes and King John
handsomely compensated for all that he lost in The Wash. Let us
thank the soaring genius of the human mind which has thus found a
means to canalize for us the waters of affliction; and let us always
be scrupulous in paying up our premiums before the date indicated
on the printed card, lest calamity should come upon us and find us
unprepared.
In a sense, though, insurance was but an empirical science until
the Indescribable Company made its appearance. The man who is
insured with the Indescribable walks the world in armour of proof;
those contrary accidents and mortifications which are a source of
spiritual profit to the saint are a source of material advantage to
him. No east wind but flatters him with the prospect of a lucrative
cold; no dropped banana skin but may suddenly hurl him into
affluence. The chicken-farmer whose hen-houses are fitted with the
company’s patent automatic egg-register can never make a failure of
his business. The egg is no sooner laid than it falls gently through a
slot which marks its passage on a kind of taximeter; and if the total
of eggs at the end of the month is below the average the company
pays—I had almost said, the company lays—an exact monetary
equivalent for the shortage. The company which thus takes upon
itself the office of a hen is equally ready when occasion arises to
masquerade as a bee: if your hives are opened in the presence of its
representative you can distend every empty cell with sweet nectar at
the company’s expense. Doctors can guarantee themselves against
an excess of panel patients, barristers against an absence of briefs.
You can insure every step you take on this side of the grave, but no
one of them on such handsome terms as the step which takes you
into the grave; and it is confidently believed that if certain practical
difficulties could be got over the Indescribable would somehow
contrive to frank your passage into the world beyond. Wags have
made merry at the company’s expense, alleging that a burglar can
insure himself against a haul of sham jewels, and a clergyman
against insufficient attendance at even-song. They tell stories of a
client who murmured “Thank God!” as he fell down a lift-shaft, and a
shipwrecked passenger who manifested the liveliest annoyance at
the promptness of his rescuers when he was being paid for floating
on a life-belt at the rate of ten pounds a minute. So thoroughly has
the Indescribable reversed our scale of values here below.
But of all the company’s enterprises none can rival in importance
or in popularity the so-called Euthanasia policy. One of the giant
brains that organized the undertaking observed with compassion the
doubtful lot of human kind, the lot which makes the business man
sweat and labour and agonize, uncertain whether he himself will
reap the fruits of his industry or whether they will pass to an heir in
whom, on the whole, he is less interested. It follows, of course, from
the actuarial point of view, that he needs a policy which covers both
possibilities, immature death or unexpected longevity, but the former
on a more princely scale than the latter. If you take out a Euthanasia
policy you will pay very heavy premiums; that goes without saying.
But you pay them with a sense of absolute security. If you should die
before the age of sixty-five a fortune is immediately distributed to
your heirs and assigns. If you outlive that crucial age you become
thenceforward, until the decree of nature takes its tardy effect, the
pensioner of the company; every faltering breath you draw in the
last stages of senility is money to you; your heirs and assigns,
instead of looking forward heartlessly to the moment of your
release, conspire to keep your body and soul together with every
known artifice of modern medicine—it is in their interest to do so.
There is but one way in which you can forfeit the manifest
advantages of the scheme, and that is self-murder. So complex is
our human fashioning that men even may be tempted to enrich their
surviving relatives by such means; and you will find, accordingly, at
the bottom of your Euthanasia policy, an ominous black hand
directing attention to the fact that in the event of suicide no benefits
are legally recoverable.
It goes without saying that the Indescribable Building is among
the finest in London. It appears to be an axiom with those who
conduct business in the modern, or American, manner that efficiency
is impossible unless all your transactions are conducted in an edifice
not much smaller and not much less elaborate than the Taj Mahal.
Why this should be so it is difficult to explain. In a less credulous age
we might have been tempted to wonder where all the money came
from; whether (to put it brutally) our premiums might not have
worked out a little lower if the company’s premises had not been
quite so high. After all, our solicitor lives in horrid, dingy little
chambers, with worn-out carpets and immemorial cobwebs on the
wall—does he never feel that this squalor will fail to inspire
confidence? Apparently not; yet the modern insurance company
must impress us all through the palatial splendour of its offices with
the idea that there is a vast reserve of capital behind it. The wildest
voluptuousness of an Eastern tyrant is less magnificent in its
architectural scheme than the hard-headed efficiency of the
American business man. Chatting in the waiting-room of some such
edifice, Sardanapalus might have protested that it stumped him how
they did it, and Kublai Khan might have registered the complaint that
it was all very well but the place didn’t feel homey.
 Indescribable House is an enormously high building with long,
narrow windows that make it look like an Egyptian tomb. It is of
white stone, of course, so time-defying in its appearance that it
seems almost blasphemous to remember the days when it was
simply a gigantic shell composed of iron girders. Over the front door
there is a group of figures in relief, more than life-size; the subject is
intended, I believe, to be Munificence wiping away the tears of
Widowhood, though the profane have identified it before now as
Uncle Sam picking Britannia’s pocket. This is continued all round the
four sides by a frieze, ingeniously calculated to remind the spectator
of the numerous risks which mortality has to run: here a motor
accident, with an ambulance carrying off the injured parties; here an
unmistakable shipwreck; there a big-game hunter being gored by a
determined-looking buffalo, while a lion prowls thoughtfully in the
background. Of the interior I cannot speak so positively, for even
those who are favoured enough to be the company’s clients never
seem to go up beyond the first storey. But rumour insists that there
is a billiard-room for the convenience of the directors (who never go
there); and that from an aeroplane, in hot weather, you can see the
clerks playing tennis on the roof. What they do when they are not
playing tennis and what possible use there can be in all those
multitudinous rooms on the fifth, sixth and seventh floors are
thoughts that paralyze the imagination.
In one of the waiting-rooms on the ground floor, sitting under a
large palm-tree and reading a closely reasoned article in the
Actuaries’ and Bottomry Gazette, sat a client to whom the reader will
do well to direct attention, for our story is concerned with him. His
look, his dress, his manner betrayed the rich man only to those who
have frequented the smaller provincial towns and know how little in
those centres money has to do with education. He had a short black
coat with very broad and long lapels, a starched collar that hesitated
between the Shakespeare and the all-the-way-and-back-again
patterns, a double-breasted waistcoat from which hung a variety of
seals, lockets and charms—in London, in fact, you would have put
him down for an old-fashioned bank cashier with a moderate
income. Actually, he could have bought you out of your present job
at double the salary and hardly felt it. In Pullford, a large Midland
town which you probably will never visit, men nudged one another
and pointed to him as one of the wealthiest residents. In the
anteroom of the Indescribable offices he looked, and perhaps felt,
like a schoolboy waiting his turn for pocket-money. Yet even here he
was a figure recognizable to the attendant who stood there
smoothing out back numbers of the Actuaries’ and Bottomry
Gazette. For this man, called Mottram by accident of birth and
Jephthah through the bad taste of his parents, was the holder of a
Euthanasia policy.
Another attendant approached him, summoning him to his
appointed interview. There was none of that “Mr. Mottram, please!”
which reverberates so grimly through the dentist’s waiting-room. At
the Indescribable the attendants come close to you and beckon you
away with confidential whispers; it is part of the tradition. Mr.
Mottram rose, and was gently sucked up by the lift to the first
storey, where fresh attendants ushered him on into one of the few
rooms that really mattered. Here he was met by a pleasant, rather
languid young man, delicately dressed, university-bred, whose
position in the complicated hierarchy of the Indescribable it is no
business of ours to determine.
“How do you do, Mr. Mottram? Keeping well, I hope?”
Mr. Mottram had the blunt manner of his fellow townsmen, and
did not appreciate the finesse of metropolitan conversational
openings. “Ah, that’s right,” he said; “best for you I should keep well,
eh? You and I won’t quarrel there. Well, it may surprise you, but it’s
my health I’ve come to talk about. I don’t look ill, do I?”
“You look fit for anything. I’d sooner be your insurance agent
than your family doctor, Mr. Mottram.” The young man was
beginning to pick up the Pullford idea of light small talk.
“Fit for anything, that’s right. And, mind you, I feel fit for
anything. Never felt better. Two years!”
“I beg your pardon?”
“Two years, that’s what he says. What’s the good of being able to
know about these things if they can’t do anything for ’em, that’s
what I want to know? And, mind you, he says there isn’t anything
for it, not in the long run. He tells me to take this and that, you
know, and give up this and that”——
“I’m sorry, Mr. Mottram, but I don’t quite understand. Is this your
doctor you’re talking about?”
“No doctor of mine. My doctor down in Pullford, he couldn’t tell
what was the matter. Sent me on to this big man in London I’ve
been seeing this morning. Two years, he says. Seems hard, doesn’t
it?”
“Oh! . . . You’ve been to a specialist. I say, I’m most awfully
sorry.” The young man was quite serious in his condolences, though
he was even more embarrassed than actually grieved. It seemed
horrible to him that this red-faced man who looked so well and
obviously enjoyed his meals should be going where Numa and Ancus
went before him: he did not fit into the picture. No taint of
professionalism entered into this immediate reaction. But Mr.
Mottram still took the business line.
“Ah! ‘sorry’—you may say that. It may mean half a million to you,
mayn’t it?”
“Yes; but, look here, these specialists are often wrong. Famous
case of one who went potty and told all his patients they were in for
it. Look here, what about seeing our man? He’d vet you, gladly.”
It need hardly be said that the Indescribable keeps its own
private physician, whose verdict must be obtained before any
important insurance is effected. He is considered to be one of the
three best doctors in England, and fantastic stories are told about
the retaining fee which induced him to give up his practice in Harley
Street. Once more the young man was entirely disinterested; once
more Mr. Mottram saw ground for suspicion. It looked to him as if
the company were determined to get stable information about the
exact state of his health, and he did not like the idea.
“It’s of no consequence, thank you all the same. It isn’t as if my
case were a doubtful one; I can give you the doctor’s certificate if
needed. But I didn’t come here to talk about that; I came on
business. You know how I stand?”
The young man had just been looking up Mr. Mottram’s docket
and knew all about him well enough. But the Indescribable cultivates
the family touch; it likes to treat its clients as man to man, not as so
many lives. “Let’s see”—the young man appeared to be dragging the
depths of memory—“you should be sixty-three now, eh? And in two
years’ time—why, it looks as if it were just touch and go whether
your policy covered a case of—h’m!—premature decease or not,
doesn’t it?”
“That’s right. My birthday’s in a fortnight’s time, more or less. If
that doctor was dead accurate, it’ll stand you in five hundred
thousand; if he put the date a bit too soon, then I get nothing, and
you pay nothing; that’s how it is, isn’t it?”
“Looks like it, I’m afraid. Of course, you’ll understand, Mr.
Mottram, the company has to work by rule of thumb in these cases.”
“I see that. But look at it this way. When I took out that policy I
wasn’t thinking much of the insurance part; I’ve no kith nor kin
except one nephew, and he’s seen fit to quarrel with me, so nothing
goes to him, anyhow. If that half-million falls in, it will just go to
charity. But what I’d set my heart on was the annuity; we’re a long-
lived family, mostly, and I’d looked forward to spending my last days
in comfort, d’you see? Well, there’s no chance of that after what the
doctor’s been telling me. So I don’t value that Youth-in-Asia policy as
much as I did, see? And I’ve come here to make you a fair offer.”
“The company”—— began the young man.
“Let me have my say, and you shall have yours afterward. They
call me rich, and I suppose I am rich; but my stuff is tied up more
than you’d think; with money as tight as it is, you can’t just sell out
of a thing when you feel inclined. What I want is ready money—
doctors’ bills, you know, and foreign travel, and treatment, and that.
So this is my offer: you pay back half the premiums from the time I
started insuring with you, half the premiums, mind you; and if I die
before I reach sixty-five, then we call it off; you pay no insurance: if
I live beyond sixty-five we call it off, and you pay no annuity. Come
now, there’s a business offer. What do you people say to it?”
“I’m sorry; I’m frightfully sorry. But, you know, we’ve had this
kind of offer before, and the company has always taken the line that
it can’t go back on the original contract. If we lose, we lose; if the
client loses, he must shoulder the responsibility. If we once went in
for cancelling our insurances like that, our whole credit would suffer.
I know you mean well by us, Mr. Mottram, and we’re grateful to you
for the generosity of the offer; but it can’t be done; really it can’t.”
There was a heavy silence for nearly a minute. Then Mr.
Mottram, pathetic in his disappointment, tried his last card.
“You could put it to the directors, couldn’t you? Stands to reason
you couldn’t accept an offer of that kind without referring it to them.
But you could put it to them at their next meeting, eh?”
“I could put it to the directors; indeed, I will. But I’m sorry to say
I can’t hold out any hopes. The premium of the Euthanasia policy is
so stiff that we’re always having people wanting to back out of it
half-way, but the directors have never consented. If you take my
advice, Mr. Mottram, you’ll take a second opinion about your health,
go carefully this next year or two, and live to enjoy that annuity—for
many years, I hope.” The young man, after all, was a paid official;
he did not stand to lose.
Mr. Mottram rose; he declined all offers of refreshment. A little
wearily, yet holding his head high, he let the confidential attendants
usher him out. The young man made some notes, and the grim
business of the Indescribable Company went on. In distant places
ships were foundering, factories were being struck by lightning,
crops were being spoiled by blight, savages were raiding the
peaceful country-side; men were lying on air-cushions, fighting for
breath in the last struggle of all. And to the Indescribable Company
all these things meant business; most of them meant loss. But the
loss never threatened its solvency for a moment; the law of
averages saw to that.
 Chapter II.
The Detective Malgré Lui
I have already mentioned that the Indescribable kept its own
tame doctor, a man at the very head of his profession. He was not in
the least necessary to it; that is to say, a far cheaper man would
have done the work equally well. But it suited the style of the
Indescribable to have the very best man, and to advertise the fact
that he had given up his practice in order to work exclusively for the
company; it was all of a piece with the huge white building, and the
frieze, and the palms in the waiting-room. It looked well. For a quite
different reason the Indescribable retained its own private detective.
This fact was not advertised; nor was he ever referred to in the
official communications of the company except as “our
representative.” He carried neither a lens nor a forceps—not even a
revolver; he took no injections; he had no stupid confidential friend;
but a private detective he was for all that. An amateur detective I
will not call him, for the company paid him, and as you would
expect, quite handsomely; but he had nothing whatever to do with
Scotland Yard, where the umbrellas go to.
He was not an ornament to the company; he fulfilled a quite
practical purpose. There are, even outside the humorous stories,
business men in a small way who find it more lucrative to burn down
their premises than to sell their stock. There are ladies—ladies
whose names the Indescribable would never dream of giving away—
who pawn their jewels, buy sham ones, and then try to make the
original insurance policy cover them in the event of theft. There are
small companies (believe it or not) which declare an annual loss by
selling their stuff below cost price to themselves under another
name. Such people flocked to the Indescribable. It was so vast a
concern that you felt no human pity about robbing it—it was like
cheating the income tax, and we all know how some people feel
about that. The Indescribable never prosecuted for fraud; instead, it
allowed a substantial margin for these depredations, which it
allowed to continue. But where shady work was suspected “our
representative” would drop in in the most natural way in the world
and by dint of some searching inquiries made while the delinquent’s
back was turned would occasionally succeed in showing up a fraud
and saving the company a few hundreds of thousands by doing so.
The company’s “representative,” and our hero, was Miles Bredon,
a big, good-humoured, slightly lethargic creature still in the early
thirties. His father had been a lawyer of moderate eminence and
success. When Miles went to school it was quite clear that he would
have to make his own way in the world, and very obscure how he
was going to do it. He was not exactly lazy, but he was the victim of
hobbies which perpetually diverted his attention. He was a really
good mathematician, for example; but as he never left a sum
unfinished and “went on to the next” his marks never did him
justice. He was a good cross-country runner, but in the middle of a
run he would usually catch sight of some distraction which made him
wander three miles out of his course and come in last. It was his
nature to be in love with the next thing he had to do, to shrink in
loathing from the mere thought of the next but one. The war came
in time to solve the problem of his career; and more fortunate than
some he managed to hit on a métier in the course of it. He became
an intelligence officer; did well, then did brilliantly; was mentioned in
despatches, though not decorated. What was more to the point, his
Colonel happened to be a friend of some minor director of the
Indescribable, and, hearing that a discreet man was needed to
undertake the duties outlined, recommended Bredon. The offer fell
at his feet just when he was demobilized; he hated the idea of it, but
was sensible enough to realize, even then, that ex-officers cannot be
choosers. He was accepted on his own terms, namely, that he
should not have to sit in an office kicking his heels; he would always
be at home, and the company might call him in when he was
wanted.
 In a few years he had made himself indispensable to his
employer; that is to say, they thought they could not get on without
him, though in fact his application to his duties was uncertain and
desultory. Four out of five inquiries meant nothing to him; he made
nothing of them; and Whitechapel thanked the God of its fathers for
his incompetence. The fifth case would appeal to his capricious
imagination; he would be prodigal of time and of pains; and he
would bring off some coup which was hymned for weeks behind
closed doors in the Indescribable Building. There was that young
fellow at Croydon, for example, who had his motor-bicycle insured,
but not his mother-in-law. Her body was found at the foot of an
embankment beside a lonely road in Kent, and there was no doubt
that it had been shot out of the side-car; only (as Bredon managed
to prove) the lady’s death had occurred on the previous day from
natural causes. There was the well-known bootlegger—well known,
at least, to the United States police—who insured all his cargoes
with the Indescribable and then laid secret information against
himself whereby vigilant officials sank hundreds of dummy cases in
the sea, all the bottles containing sea-water. And there was the lady
of fashion who burgled her own jewels in the most plausible manner
you could imagine and had them sold in Paris. These crooked ways
too the fitful intuitions of Miles Bredon made plain in the proper
quarters.
He was well thought of, in fact, by every one except himself. For
himself, he bitterly regretted the necessity that had made him
become a spy—he would use no other word for it—and constantly
alarmed his friends by announcing his intention of going into the
publishing trade, or doing something relatively honest. The influence
which saved him on these occasions was that of—how shall I say it?
—his wife. I know—I know it is quite wrong to have your detective
married until the last chapter, but it is not my fault. It is the fault of
two mocking eyes and two very capable hands that were employed
in driving brass-hats to and fro in London at the end of the war.
Bredon surrendered to these, and made a hasty but singularly
fortunate marriage. Angela Bredon was under no illusions about the
splendid figure in khaki that stood beside her at the altar. Wiser than
her generation, she realized that marriages were not “for the
duration”; that she would have to spend the rest of her life with a
large, untidy, absent-minded man who would frequently forget that
she was in the room. She saw that he needed above all things a
nurse and a chauffeur, and she knew that she could supply both
these deficiencies admirably. She took him as a husband, with all a
husband’s failings, and the Indescribable itself could not have
guaranteed her more surely against the future.
There is a story of some Bishop, or important person, who got
his way at Rome rather unexpectedly over an appeal, and, when
asked by his friends how he did it, replied, “Fallendo infallibilem.” It
might have been the motto of Angela’s mastery over her husband;
the detective, always awake to the possibilities of fraudulent dealing
in every other human creature, did not realize that his wife was a
tiny bit cleverer than he was and was always conspiring for his
happiness behind his back. For instance, it was his custom of an
evening to play a very long and complicated game of patience,
which he had invented for himself; you had to use four packs, and
the possible permutations of it were almost unlimited. It was an
understood thing in the household that Angela, although she had
grasped the rules of the game, did not really know how to play it.
But when, as often happened, the unfinished game had to be left
undisturbed all night, she was quite capable of stealing down early
in the morning and altering the positions of one or two cards, so
that he should get the game “out” in time to cope with his ordinary
work. These pious deceits of hers were never, I am glad to say,
unmasked.
About a fortnight after Mr. Mottram’s interview with the young
man at Indescribable House these two fortunate people were alone
together after dinner, she alternately darning socks and scratching
the back of a sentimental-looking fox-terrier, he playing his
interminable patience. The bulk of the pack lay on a wide table in
front of him, but there were outlying sections of the design dotted
here and there on the floor within reach of his hand. The telephone
bell rang, and he looked up at her appealingly—obviously, he was
tied hand and foot by his occupation—which to her only meant
putting her darning away, lifting the fox-terrier off her feet, and
going out into the hall. She understood the signal, and obeyed it.
There was a fixed law of the household that if she answered a call
which was meant for him he must try to guess what it was about
before she told him. This was good for him, she said; it developed
the sleuth instinct.
“Hullo! Mrs. Bredon speaking—who is it, please? . . . Oh, it’s
you. . . . Yes, he’s in, but he’s not answering the telephone. . . . No,
only drunk. . . . Just rather drunk. . . . Business? Good; that’s just
what he wants. . . . A man called what? . . . M-o-t-t-r-a-m, Mottram,
yes. . . . Never heard of it. . . . St. William’s? Oh, the Midlands, that
are sodden and unkind, that sort of Midlands, yes? . . . Oh! . . . Is it
—what? . . . Is it supposed to have been an accident? . . . Oh, that
generally means suicide, doesn’t it? . . . Staying where? . . . Where’s
that? . . . All right, doesn’t matter; I’ll look it up. . . . At an inn? Oh,
then it was in somebody else’s bed really! What name? . . . What a
jolly name! Well, where’s Miles to go? To Chilthorpe? . . . Yes, rather,
we can start bright and early. Is it an important case? Is it an
important case? . . . Oo! I say! I wish I could get Miles to die and
leave me half a million! Righto, he’ll wire you to-morrow. . . . Yes,
quite; thanks. . . . Good-night.”
“Interpret, please,” said Angela, returning to the drawing-room.
“Why, you’ve been going on with your patience the whole time! I
suppose you didn’t listen to a word I was saying?”
“How often am I to tell you that the memory and the attention
function inversely? I remember all you said, precisely because I
wasn’t paying attention to it. First of all, it was Sholto, because he
was ringing you up on business, but it was somebody you know
quite well—at least I hope you don’t talk like that to the tradesmen.”
“Sholto, yes, ringing up from the office. He wanted to talk to
you.”
“So I gathered. Was it quite necessary to tell him I was drunk?”
“Well, I couldn’t think of anything else to say at the moment. I
couldn’t tell him you were playing patience, or he might have
thought we were unhappily married. Go on, Sherlock.”
 “Mottram, living at some place in the Midlands you’ve never
heard of, but staying at a place called ‘Chilthorpe’—he’s died, and his
death wants investigating; that’s obvious.”
“How did you know he was dead?”
“From the way you said ‘Oh’—besides, you said he’d died in his
bed, or implied it. And there’s some question of half a million
insurance—Euthanasia, I suppose? Really, the Euthanasia’s been
responsible for more crimes than psychoanalysis.”
“Yes, I’m afraid you’ve got it all right. What did he die of?”
“Something that generally means suicide—or rather, you think it
does. The old sleeping draught business? Veronal?”
“No stupid, gas. The gas left turned on. And where’s Chilthorpe,
please?”
“It’s on the railway. If my memory serves me right, it is
Chilthorpe and Gorrington, between Bull’s Cross and Lowgill
Junction. But the man, you say, belongs somewhere else?”
“Pullford; at least it sounded like that. In the Midlands
somewhere, he said.”
“Pullford, good Lord, yes. One of these frightful holes. They make
perambulators or something there, don’t they? A day’s run, I should
think, in the car. But of course it’s this Chilthorpe place we want to
get to. You wouldn’t like to look it up in the gazetteer while I just get
this row finished, would you?”
“I shan’t get your sock finished, then. On your own foot be it!
Let’s see, here’s Pullford all right. . . . It isn’t perambulators they
make, it’s drain-pipes. There’s a grammar school there, and an
asylum; and the parish church is a fine specimen of early Perp.,
extensively restored in 1842; they always are. Has been the seat of
a Roman Catholic Bishopric since 1850. The Baptist Chapel”——
“I did mention, didn’t I, that it was Chilthorpe I wanted to know
about?”
“All in good time. Let’s see, Chilthorpe—it isn’t a village really, it’s
a ship town. It has 2,500 inhabitants. There’s a lot here about the
glebe. It stands on the River Busk, and there is trout fishing.”
“Ah, that sounds better.”
“Meaning exactly?”
 “Well, it sounds as if the fellow had done himself in by accident
all right. He went there to fish—you don’t go to a strange village to
commit suicide.”
“Unless you’ve got electric light in your house and want to
commit suicide with gas.”
“That’s true. What was the name of the inn, by the way?”
“The Load of Mischief. Such a jolly dedication, I think.”
“Now let’s try the map.”
“I was coming to that. Here’s the Busk all right. I say, how funny,
there’s a place on the Busk called ‘Mottram.’ ”
“Anywhere near Chilthorpe?”
“I haven’t found it yet. Oh, yes, here it is, about four miles away.
Incidentally, it’s only twenty miles or so from Pullford. Well, what
about it? Are we going by car?”
“Why not? The Rolls is in excellent condition. Two or three days
ought to see us through; we can stay, with any luck, at the Load of
Mischief, and the youthful Francis will be all the better for being left
to his nurse for a day or two. You’ve been feeding him corn, and he
is becoming obstreperous.”
“You don’t deserve to have a son. However, I think you’re right. I
don’t want to trust you alone in a ship town of 2,500 inhabitants,
some of them female. Miles, dear, this is going to be one of your big
successes, isn’t it?”
“On the contrary, I shall lose no time in reporting to the directors
that the deceased gentleman had an unfortunate accident with the
gas, and they had better pay up like sportsmen. I shall further point
out that it is a great waste of their money keeping a private spy at
all.”
“Good, then I’ll divorce you! I’m going to bed now. Not beyond
the end of that second row, mind; we shall have to make an early
start to-morrow.”
 Chapter III.
At the Load of Mischief
By next morning Bredon’s spirits had risen. He had received by
the early post a confidential letter from the company describing Mr.
Mottram’s curious offer, and suggesting (naturally) that the state of
his health made suicide a plausible conjecture. The morning was
fine, the car running well, the road they had selected in admirable
condition. It was still before tea time when they turned off from its
excellent surface onto indifferent by-roads, through which they had
to thread their way with difficulty. The signposts, as is the wont of
English signposts, now blazoned “Chilthorpe,” “Chilthorpe,”
“Chilthorpe,” as if it were the lodestone of the neighbourhood, now
passed it over in severe silence, preferring to call attention to the
fact that you were within five furlongs of Little Stubley. They had
fallen, besides, upon hill country, with unexpected turns and
precipitous gradients; they followed with enforced windings the
bleak valley of the Busk, which swirled beneath them over smooth
boulders between desolate banks. It was just after they had refused
the fifth invitation to Little Stubley that the County Council’s
arrangements played them false; there was a clear issue between
two rival roads, with no trace of a signpost to direct their preference.
It was here that they saw, and hailed, an old gentleman who was
making casts into a promising pool about twenty yards away.
“Chilthorpe?” said the old gentleman. “All the world seems to be
coming to Chilthorpe. The County Council does not appear to have
allowed for the possibility of its becoming such a centre of fashion. If
you are fond of scenery, you should take the road to the left; it goes
over the hill. If you like your tea weak, you had better take the
valley road to the right. Five o’clock is tea time at the Load of
Mischief, and there is no second brew.”
Something in the old gentleman’s tone seemed to invite
confidences. “Thank you very much,” said Bredon. “I suppose the
Load of Mischief is the only inn that one can stop at?”
“There was never much to be said for the Swan. But to-day the
Load of Mischief has added to its attractions; it is not everywhere
you can sleep with the corpse of a suicide in the next room. And the
police are in the house, to satisfy the most morbid imagination.”
“The police? When did they come?”
“About luncheon time. They are understood to have a clue. I am
only afraid, myself, that they will want to drag the river. The police
always drag the river if they can think of nothing else to do.”
“You’re staying at the inn, I gather?”
“I am the surviving guest. When you have tasted the coffee in
the morning you will understand the temptation to suicide; but so
far I have resisted it. You are not relatives, I hope, of the
deceased?”
“No; I’m from the Indescribable. We insured him, you know.”
“It must be a privilege to die under such auspices. But I am
afraid I have gone beyond my book: when I say poor Mottram
committed suicide I am giving you theory not fact.”
“The police theory?”
“Hardly. I left before they arrived. It is the landlady’s theory, and
when you know her better you will know that it is as well not to
disagree with her; it provokes discussion.”
“I am afraid she must be very much worried by all this.”
“She is in the seventh heaven of lamentation. You could knock
her down, she tells me, with a feather. She insists that her custom is
ruined for ever; actually, you are the second party to stay at the inn
as the result of this affair, and the jug and bottle business at mid-day
was something incredible. The Band of Hope was there en masse,
swilling beer in the hope of picking up some gossip.”
“The other party, were they relations?”
“Oh no, it’s a policeman; a real policeman from London. The
secretary, I suppose, must have lost his head, and insisted on
making a cause célèbre of the thing. I forgot him, by the way, a little
chap called Brinkman; he’s at the Load too. A thousand pardons, but
I see a fish rising. It is so rare an event here that I must go and
attend to it.” And, nodding pleasantly, the old gentleman made his
way to the bank again.
Chilthorpe is a long, straggling village with the business part
(such as it is) at the lower end. The church is here, and the Load of
Mischief, and a few shops; here, too, the Busk flows under a wide
stone bridge—a performance which at most times of the day attracts
a fair crowd of local spectators. The houses are of grey stone, the
roofs of blue slate. The rest of the village climbs up along the valley
all in one street; the houses stand perched on the edge of a steep
slope, too steep almost for the cultivation of gardens, though a few
currant and gooseberry bushes retain a precarious foothold. The
view has its charms; when mists hang over it in autumn, or when
the smoke of the chimneys lingers idly on a still summer evening, it
has a mysterious and strangely un-English aspect.
The hostess, presumably to be identified with “J. Davis, licensed
to sell wines, spirits and tobacco,” met them on the threshold,
voluble and apparently discouraging. Her idea seemed to be that she
could not have any more guests coming and committing suicide in
her house. Bredon, afraid that his patience or his gravity would
break down, put Angela in charge of the conversation, and so
delicate was her tact, so well-placed her sympathy, that within ten
minutes their arrival was being hailed as a godsend, and Mrs. Davis,
ordering the barmaid to bring tea as soon as it could be procured,
ushered them into a private room, assuring them of accommodation
upstairs when she could put things to rights. It had been one thing
after another, she complained, all day, she didn’t really hardly know
which way to turn, and her house always a respectable one. There
was not much custom, it seemed, at Chilthorpe, lying so far away
from the main road and that—you would have supposed that in a
R.A.C. Listed Hotel suicides were a matter of daily occurrence, and
the management knew how to deal with them. Whereas Mrs. Davis
hadn’t anybody but the girl and the Boots, and him only with one
arm. And those boys coming and looking in through the front
window; “disgraceful,” she called it; and what were the police for if
they couldn’t put a stop to it? And the reporters—six of them she’d
turned away that very day—coming and prying into what didn’t
concern them. They didn’t get a word out of her, that was one thing.
Though, mark you, if Mrs. Davis didn’t know poor Mr. Mottram,
who did? Coming there regular year after year for the fishing, poor
gentleman; such a quiet gentleman too, and never any goings-on.
And how was she to know what would come of it? It wasn’t that the
gas leaked; time and again she’d had those pipes seen to, and no
complaints made. If there had have been anything wrong, Mr.
Pulteney, he’d have let her hear about it, he was one for having
everything just as he liked, and no mistake. . . . Yes, that would be
him, he was a great one for the fishing. Such a queer gentleman
too, and always taking you up short. Why, yesterday morning, when
she went to tell him about what had happened in the night he was
as cool as anything; all he said was, “In that case, Mrs. Davis, I will
fish the Long Pool this morning,” like that he said. Whereas Mr.
Brinkman, that was the secretary, he was in a great taking about it,
didn’t hardly know what he said or did, Mr. Brinkman didn’t. And to
think of all the gas that was wasted; on all night it was, and who
was to pay for it was more than she knew. Summing up, Mrs. Davis
was understood to observe that it was a world for sorrow, and man
was cut down like a flower, as the sparks fly upward. However, there
was them above as knew, and what would be would be.
Of all this diatribe Bredon was a somewhat languid auditor. He
recognized the type too well to suppose that any end was to be
gained by cross-examination. Angela cooed and sighed, and dabbed
her eyes now and again at appropriate moments, and in so doing
won golden opinions from the tyrannous conversationalist. It was a
strong contrast when the maid came in with the tea things; she
plumped them down in silence, tossing her head defiantly, as who
should imply that somebody had recently found fault with her behind
the scenes, but she was not going to take any notice of it. She was a
strapping girl, of undeniable good looks, spoilt (improved, the Latins
would have said) by a slight cast in one eye. In the absence of any
very formidable competition it was easy to imagine her the belle of
the village. So resolute did her taciturnity appear that even Angela,
who could draw confidences from a stone, instinctively decided that
it would be best to question her later on. Instead, she whiled away
the interminable interval which separates the arrival of the milk jug
from that of the teapot by idly turning over the leaves of the old-
fashioned visitors’ book. The Misses Harrison, it appeared, had
received “every attention” from their kind and considerate hostess.
The Pullford Cycling Club had met for its annual outing, and the
members pronounced themselves “full to bursting, and coming back
next year.” An obviously newly married couple had found the
neighbourhood “very quiet”; a subsequent annotator had added the
words “I don’t think!!!” with the three marks of exclamation. The
Wotherspoon family, a large one, testified to having had a “rattling
good time” at this old-world hostelry. The Reverend Arthur and Mrs.
Stump would carry away “many pleasant memories” of Chilthorpe
and its neighbourhood.
Miles was wandering aimlessly about the room inspecting those
art treasures which stamp, invariably and unmistakably, the best
room of a small country inn. There was the piano, badly out of tune,
with a promiscuous heap of dissenting hymn-books and forgotten
dance tunes reposing on it. There were the two pictures which
represent a lovers’ quarrel and a lovers’ reconciliation, the hero and
heroine being portrayed in riding costume. There was a small
bookshelf, full of Sunday-school prizes, interspersed with one or two
advanced novels in cheap editions, clearly left behind by earlier
visitors. There was a picture of Bournemouth in a frame of repulsive
shells. There was a photograph of some local squire or other on
horseback. There were several portraits which were intended to
perpetuate the memory of the late Mr. Davis, a man of full bodily
habit, whose clothes, especially his collar, seemed too tight for him.
There were a couple of young gentlemen in khaki on the
mantelpiece; there was a sailor, probably the one who had collected
the strange assortment of picture post-cards in the album under the
occasional table; there were three wedding groups, all apparently in
the family—in a few words, a detective interested in such problems
might have read there, a picture, the incredibly long and complicated
annals of the poor.
To Bredon it was all a matter of intense irritation. When he
visited the scene of some crime or some problem, he was fond of
poking his way round the furniture, trying to pick up hints from the
books and the knickknacks about the character of the people he was
dealing with. At least, he would say, if you cannot pick up evidence
about them you can always catch something of their atmosphere.
Mottram had hardly played the game when he died in a country inn
where he had not been able to impress his surroundings with any
touch of his own quality; this inn parlour was like any other inn
parlour, and the dead body upstairs would be a problem in isolation,
torn away as it was from its proper context. The bedroom doubtless
would have a text over the washing-stand, a large wardrobe stuffed
with family clothes and mothballs, a cheap print of the “Soul’s
Awakening”; it would just be an inn bedroom, there would be no
Mottram about it.
“I say,” Angela interrupted suddenly, “Mottram seems to have
visited this place pretty regularly, and always for the fishing season.
There are some fine specimens of his signature; the last only written
two days ago.”
“Eh? What’s that?” said Bredon. “Written his signature in already,
had he? Any date to it?”
“Yes, here it is, ‘J. W. Mottram, June 13th to’—and then a blank.
He didn’t know quite how long he would be staying, I suppose.”
“Let’s see. . . . Look here, that’s all wrong, you know. This isn’t a
hotel register; it’s just a visitors’ book. And people who write in a
visitors’ book don’t write till the day they leave.”
“Necessarily?”
“Invariably. Look here: look at Arthur Stump. You can see from
his style and his handwriting what a meticulous fellow he is. Well, he
came here on May twenty-first, and stayed till May twenty-six. The
Wilkinsons came here a day later, on the twenty-second, and left on
the twenty-fourth. But the Wilkinson entry comes first, and that’s
because they left first, don’t you see? And here is Violet Harris doing
the same; she puts her name before the Sandeman party. Look at