Low-Temperature Microscopy and Analysis

553 Pages·1992·16.158 MB·English

by  Patrick Echlin (auth.)

Low-Temperature Microscopy
and Analysis
Low-Temperature Microscopy
and Analysis
Patrick Echlin
University of Cambridge
Cambridge, England
Springer Science+Business Media, LLC
Library of Congress Cataloging-in-Publication Data
Echiin , Parr ick .
Low-temperature microscopy and analysis / Patrick Echlin.
p. cm.
Includes bibliographical references and index.
1. Cryomicroscopy. 2. Cryopreservation of organs, tissues, etc.
I. Title.
QH225.E34 1992
578'.4—dc20 91-39738
CIP
Figures 5.11, 9.4, 9.6, and 9.7 from Journal of Electron Microscopy Technique, reprinted by
permission of John Wiley and Sons, Inc.
ISBN 978-1-4899-2304-2 ISBN 978-1-4899-2302-8 (eBook)
DOI 10.1007/978-1-4899-2302-8
© Springer Science+Business Media New York 1992
Originally published by Plenum Press, New York in 1992
Softcover reprint of the hardcover 1st edition 1992
All rights reserved
No part of this book may be reproduced, stored in a retrieval system, or transmitted
in any form or by any means, electronic, mechanical, photocopying, microfilming,
recording, or otherwise, without written permission from the Publisher
To my wife, Shirley
Very high and very low temperatures extinguish all human sympathy
and relations. It is impossible to feel affection beyond 780 or below 200
of Fahrenheit: human nature is too solid or too liquid beyond these
limits
-SYDNEY SMITH, 1836
I have gathered a posie of other men's flowers and nothing but the
thread that binds them is my own.
-MICHEL MONTAIGNE, 1592
Foreword
The frozen-hydrated specimen is the principal element that unifies the subject of low
temperature microscopy, and frozen-hydrated specimens are what this book is all
about. Freezing the sample as quickly as possible and then further preparing the
specimen for microscopy or microanalysis, whether still embedded in ice or not:
there seem to be as many variations on this theme as there are creative scientists with
problems of structure and composition to investigate. Yet all share a body of com
mon fact and theory upon which their work must be based. Low-Temperature Micros
copy and Analysis provides, for the first time, a comprehensive treatment of all the
elements to which one needs access.
What is the appeal behind the use of frozen-hydrated specimens for biological
electron microscopy, and why is it so important that such a book should now have
been written? If one cannot observe dynamic events as they are in progress, rapid
specimen freezing at least offers the possibility to trap structures, organelles, macro
molecules, or ions and other solutes in a form that is identical to what the native
structure was like at the moment of trapping. The pursuit of this ideal becomes all
the more necessary in electron microscopy because of the enormous increase in
resolution that is available with electron-optical instruments, compared to light
optical microscopes. On the size scale below one micrometer, frozen-hydrated speci
mens offer the hope of escaping from the dilemma that the "unlimited" resolution
of electron optics can, on the one hand, often be wasted by inadequate specimen
preparation, while light microscopy can give perfect specimen preparation, but only
inadequate resolution. In this context, the time has certainly come in which a compre
hensive and unified coverage of low-temperature techniques can strongly influence
the continued development of biological electron microscopy and microanalysis.
The pursuit of improved, if not ideal specimen preparation by low-temperature
techniques has developed steadily over more than 25 years. Methods have been
developed at the level of cellular fine structure (notably freeze fracture and freeze
substitution), microanalysis of diffusible substances, the structure of macromolecular
assemblies (notably freeze-drying and shadowing), and most recently even the inter
nal structure of macromolecules. At the highest resolution, low temperature is needed
not so much to preserve the native, hydrated state, which it does admirably well (but
so do other techniques, such as glucose embedment), as for the extra margin of
protection which it provides against radiation damage.
The development of all of these techniques has been aided not just a little in the
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x FOREWORD
past by the author of this book, Patrick Echlin, through his effort in organizing a
series of four International Meetings on Low-Temperature Microscopy, beginning
in 1977, and through the emphasis that he has given to the field in his role as
editor of the Journal of Microscopy. The next logical step, given the maturity of
development of the subject, would have to be nothing else than to write this book.
It is a volume that will speed access to existing techniques and greatly expand aware
ness of related work for all who seek a unified presentation of low-temperature
methods and their underlying theoretical foundation. Publication of this book there
fore makes a truly important contribution toward advancing the process of learning
what goes on in biology at a level below what can be seen with the light microscope.
Robert M. Glaeser
Department of Molecular and Cell Biology
Universitv of California at Berkelev
Preface
Water is the most abundant and most important molecule in the biosphere and outer
lithosphere. As a vapor it forms a vital envelope around our planet; as a liquid it
covers about 75% of the Earth's surface and dissolves almost everything. As a solid
it is permanently present at the Poles and on many mountain peaks and is a seasonal
reminder of the changing climate of our environment. Liquid water is vital for living
organisms. It is both a reactant and the medium in which reactions occur and their
products are transported. Water is the most abundant and least expensive building
block of living matter and when converted to the solid state can provide the perfect
matrix in which to study the structure and in situ chemistry of hydrated material.
This book considers the nature of this solid matrix, its constituent components, and
how it may be formed, manipulated, examined, and analyzed.
This volume has grown from the firm belief that low-temperature microscopy
and analysis is the only way we may hope to obtain a true picture of the fine structure
and composition of ourselves and our water-filled environment. I will discuss the
physical basis and the practical aspects of the different procedures we need to use,
the problems that occur, and the advantages that accrue. The conversion of liquids
(primarily water) to their solid phases (primarily ice) forms a central feature of this
book. The text falls into four unequal parts. The first three chapters consider water
in the liquid and solid states. The next four chapters discuss the various manipula
tions we may make to the solidified matrix. There then follows three chapters that
show what we may hope to see in the frozen samples by means of photons and
electrons, and another chapter considers the processes we need to use to analyze
their constituent elements and molecules. The final chapter contains updated infor
mation on the whole subject.
The book provides a number of well-tested procedures that will enable the
novice to cryomicroscopy and analysis to get started. It also gives a detailed back
ground from which future developments can take place. The reader is provided with
sufficient general information on how to implement a particular low-temperature
process and the reasons why it should be used. A comprehensive bibliography at the
end of the book provides the provenance and specific details of existing practices.
Low-temperature microscopy and analysis is not the sole preserve of biologists and
those interested in hydrated organic samples-although these types of samples pre
sent both the greatest challenge to the existing technologies and the only hope of
xi
xii PREFACE
solving the unresolved questions posed by such samples. The processes that will be
discussed can be used to study and analyze the solid state of all the liquid and gaseous
materials which exist on our planet (with the possible exception of helium). Low
temperatures provide an important way to study radiation-sensitive and labile sam
ples, hydrated organic systems, and phenomena that only exist at temperatures at
which living processes stop.
Low-temperature microscopy and analysis is not without potential dangers, and
it is important that experimentalists are fully aware of safety issues in the laboratory.
Cryogenic liquids can cause severe bums, and exposed parts of the body must be
protected with the appropriate clothing and face masks when using these materials.
Liquid nitrogen should only be used in a well-ventilated laboratory, as I liter of the
liquid expands to nearly 700 liters of an inert tasteless gas which can cause asphyxia.
Some secondary organic liquid cryogens have very low flash points and form danger
ously explosive mixtures with oxygen, which may condense from the atmosphere.
Some of the resins used in low-temperature embedding may cause contact dermatitis
and, like all laboratory chemicals, should be handled with gloves. An electron micro
scope laboratory is replete with potential hazards associated with vacuum and high
pressure equipment and containers, high voltages and ionizing radiation, and toxic
and inflammable chemicals. Reputable industrial companies provide safety informa
tion about their products and supplies; responsible governments issue safety legisla
tion about laboratory practices. These warnings should be heeded, because by
understanding the potential difficulties and adopting sensible laboratory procedures,
low-temperature microscopy and analysis can take place in a safe, productive (and
happy) environment.
The arguments for adopting cryotechniques and low-temperature microscopy
and analysis are secure and proven. It should come as no surprise that so many
people are now using one or more of these methods to examine and analyze hydrated,
liquid, and gaseous specimens. It is, however, astonishing that in 1991, the one
hundredth anniversary of the electron, anyone should continue to use an electron
beam instrument at ambient temperatures.
Patrick Echlin
Cambridge