Table Of ContentINTERNATIONAL SERIES IN PURE AND APPLIED BIOLOGY
Zoology Division: Volume 55
General Editor: G. A. KERKUT
SOME OTHER TITLES OF INTEREST IN THE
PERGAMON INTERNATIONAL LIBRARY
BACQ Fundamentals of Biochemical Pharmacology
BACQ and ALEXANDER Fundamentals of Radiobiology
BELFIELD and DEARDEN A Practical Course in Biology
BIRCH et al. Food Science
CAMPBELL The Structure and Functions of Animal Cell Components
CEMBER Introduction to Health Physics
CLEGG General Science: Biology
COHEN Living Embryos
CREW The Foundations of Genetics
DAVIS and BOLAN Physical Diagnosis in Medicine
DEARDEN and DEARDEN A Modern Course in Biology
FAHN Plant Anatomy
GOODWIN and MERCER Introduction to Plant Biochemistry
GOSS Physiology of Plants and their Cells
INGLIS Human Biology
JAMIESON and REYNOLDS Tropical Plant Types
KENT The Technology of Cereals
KOREN Environmental Health and Safety
LAMB and HARDEN The Meaning of Human Nutrition
LAWRIE Meat Science
OLIVER Principles of the Use of Radioisotope Tracers in Clinical and Researcl
Investigations
PANTELOURIS Introduction to Animal Physiology and Physiological Genetics
PARKE The Biochemistry of Foreign Compounds
PARSONS and TAKAHASHI Biological Oceanographic Processes
PERCIVAL Floral Biology
PINNIGER Jones' Animal Nursing
RHODES and FLETCHER Principles of Industrial Microbiology
ROLLS The Brain and Reward
STREET and COCKBURN Plant Metabolism
WAREING and PHILLIPS The Control of Growth and Differentiation in Plants
WHITE and THORSON The Kinetics of Muscle Contraction
WISCHNITZER Introduction to Electron Microscopy
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THE ULTRASTRUCTURE
OF THE
ANIMAL CELL
SECOND EDITION
BY
L. T. THREADGOLD
DEPARTMENT OF ZOOLOGY, QUEEN'S UNIVERSITY, BELFAST
PERGAMON PRESS
OXFORD NEW YORK TORONTO
SYDNEY · PARIS · FRANKFURT
U. K. Pergamon Press Ltd., Headington Hill Hall, Oxford
OX3 OBW, England
U. S. A. Pergamon Press Inc., Maxwell House, Fairview Park,
Elmsford, New York 10523, U.S.A.
CANADA Pergamon of Canada Ltd., P.O. Box 9600, Don Mills
M3C 2T9, Ontario, Canada
AUSTRALIA Pergamon Press (Aust.) Pty. Ltd., 19a Boundary
Street, Rushcutters Bay, N.S.W. 2011, Australia
FRANCE Pergamon Press SARL, 24 rue des Ecoles,
75240 Paris, Cedex 05, France
WEST GERMANY Pergamon Press GmbH, 6242 Kronberg-Taunus,
Pferdstrasse 1, Frankfurt-am-Main, West Germany
Copyright© 1976 Pergamon Press
All Rights Reserved. No part of this publication may be
reproduced, stored in a retrieval system or transmitted in
any form or by any means: electronic, electrostatic,
magnetic tape, mechanical, photocopying, recording or
otherwise, without permission in writing from the
publishers
First edition 1967
Reprinted 1969
Second edition 1976
Library of Congress Cataloging in Publication Data
Threadgold, L. T.
The Ultrastructure of the animal cell.
(International series in pure and applied biology; v. 55)
1. Cytology. 2. Ultrastructure (Biology)
I. Title
QH581.2.T49 1975 591.8'72 75-17560
ISBN 0-08-018958-X
ISBN 0-08-018957-1 flexicover
Printed in Great Britain by A. Wheaton & Co., Exeter
To the late Professor J. Bronte Gatenby, a great cytologist, who first
showed me the limitless fascination of the cell, and who would have
revelled in the exciting discoveries of modern cytology.
FOREWORD
"Yes, I have a pair of eyes," replied Sam, "and that's just it. If they wos a pair o' patent
double million magnifyin' gas microscopes of hextra power, p'haps I might be able to see
through a flight o' stairs or a deal door; but bein' only eyes, you see, my vision's limited."
(Sam Weller in Pickwick Papers, CHARLES DICKENS)
Of necessity this second edition has been extensively rewritten, revised, and
reorganized, because the alarming rate of growth in the knowledge of cell ultrastructure,
referred to in the foreword of the first edition, has gone on apace. Despite these ex
tensive changes, however, the philosophy behind the first edition has been retained. This
is essentially a text concerned with the morphology and functional morphology of cells
carried down to the level of molecular structure wherever feasible. Purely chemical, bio
chemical, and physiological aspects of cells have continued to be intentionally neglected,
so as to make the subject manageable and readable, while giving adequate depth and
cover. The reader must himself be the one who integrates or synthesizes the many aspects
of cells into a coherent whole.
The philosophy and approach outlined above may not appeal to all cell biologists, but
it still seems valid to me to select consciously one aspect of the cell and to treat it alone,
even though I realize the inherent limitations involved. The favourable response to the
first edition suggests that both students and teachers found the approach and text to their
liking and this has encouraged and confirmed my decision not to alter the essential
organization of the book.
Nevertheless, the reader must be warned and cautioned at this point, because a text
based on researches aimed at integrating form and function, as this one is, is open to
misunderstanding if not read with the correct attitude. Basically, two different types of
statements can be made concerning electron micrographs. The first type is a simple
statement of observation relating to size, shape, position, or other morphological factors
and can be verified by any observer viewing the same image; it is an objective statement.
The second type is a statement of interpretation, that is the observer's conclusions or
speculations regarding the possible or probable function of the structure observed, either
in isolation or in relation to other or ganoids, or functions, or to the structure and
function of the cell as a whole; this is a subjective statement and may vary considerably
depending on the viewer, even if information regarding the function of that structure is
available from other fields, e.g. biochemistry or physiology.
In this text I have tried to keep these two types of statements clearly differentiated.
The observations are written as simple statements, e.g. structure A is membrane bound, or
the granules in structure B are hexagonally packed, whereas interpretative or speculative
statements are preceded by qualifying clauses, e.g. it has been suggested that, it is
presumed that, or it is proposed that, etc. The reader must be conscious of the
distinction between these two types of statements. The first can be accepted with little or
IX
Foreword
no reservation, provided the reader accepts that the image observed is the result of
applying a series of techniques to a previously living and dynamic system. The latter type
of statement must always be treated with reservation, the degree of which will vary with
the comprehensiveness of the evidence and the experience and judgement of the reader.
Although interpretative and speculative statements must be critically evaluated and treated
with caution, it does not follow that they should never be made or quoted. Such inter
pretations can be tested by further observations or experiments, and are indeed the
stimulus and spark for further research. Their value lies in this and, as pointed out in the
foreword to the first edition, without speculation, observations incompletely serve their
purpose and fail to pave the way for the redirection of research further along old or into
new pathways.
As in the first edition, I should like to thank colleagues within the department for
help, advice, and technical assistance. It is also a pleasure once again to thank most
sincerely all those researchers who have generously supplied new electron micrographs and
other illustrative material; they have treated me most generously. All have been
acknowledged beneath the appropriate illustrations and I hope this will again be accepted
as an adequate expression of my appreciation.
Finally, I wish to thank once again my wife, Jenny, not only for willingly
undertaking typing and other chores, which have greatly lightened my load, but also for
constant interest and encouragement throughout a difficult task.
x
INTRODUCTION TO
ELECTRON MICROSCOPY
This short introduction to the principles and techniques of electron microscopy is for those
readers unfamiliar with the method. It was felt that this introduction was necessary in
order that such readers might appreciate both the advantages and limitations of the
technique, for it is only with an informed appreciation that a reader can make his own
critical evaluation of electron microscope studies and their results.
Image Formation
The electron microscope is based on the response of electrons (negatively charged
particles) to electromagnetic fields. A metal filament heated in a vacuum emits electrons
which can be accelerated by a difference in electrical potential between the filament, at
40—100 kV and the anode, which is positive and at zero potential (Figs. I.la and 1.2).
The resulting stream of electrons follows a straight path and has properties similar to
those of light, i.e. both corpuscular and wave characteristics at the same time. The beam
can, therefore, be focused by means of electromagnetic lenses (Figs. Lib and 1.2). The
lenses are coils of wire enclosed in a soft iron casing. When a current flows in the coil, a
magnetic field is set up at right angles to the direction of the electron beam. In order to
increase and concentrate the magnetic field without excessive increase in current flow, a
narrow annular gap is left in the iron casing facing the cavity of the coil and a soft iron
pole piece inserted to reduce the diameter of the cavity (Fig. Lib).
The electron microscope is, therefore, analogous with the light microscope (Fig. 1.2).
The "light" source is the heated filament which emits a beam of electrons (thermionic
emission). This beam is focused on the plane of the object by means of a wire coil
(magnetic lens) which acts as a condenser lens. After passing through the object, the beam
is deflected by a further magnetic lens which acts as an objective lens. The resulting
enlarged image is passed through another lens, the projector or ocular lens, which throws
the final magnified image on to a fluorescent screen. A photographic plate may be
substituted for the fluorescent screen and exposed. Such photographs are the only
permanent record available.
The analogy between a light and electron microscope is not absolute, however, and
the two instruments differ in certain essential respects. Although electromagnetic lenses are
subject to both spherical and chromatic aberration, as are optical lenses, they do not have a
fixed focal length. In electromagnetic lenses the focal length depends on the strength of
the magnetic field, which can be varied by alteration of the current passing through the
lens coil.
XI
Introduction to Electron Microscopy
Spherical and chromatic aberration of the magnetic lenses are due to either inherent
asymmetrical fields which the lenses form, or to the additional distortion of the field as
the result of the lens apertures becoming contaminated by dirt. Lens apertures are metal
plates, with holes varying from 25 to 100 μιτι, which are placed in the bore of the
condenser and objective lenses; they increase the contrast of the image by reducing the
aperture of the lens.
^Heater current
- High voltage
R = Bias resistor
(a)
-Cathode shield
Filament = Cathode
Equipotentials of
the field =
converging lens
Anode
Pole piece
-Annular gap
Solenoid winding
(b)
-Soft iron casing
Electron path
FIG. 1.1. (a) Diagram of a self-biasing electron gun. Electrons produced by the heated filament
are accelerated through the gap in the anode by the potential difference (40 to 100 kV)
between the cathode and anode. (Original.) (b) Diagram of an electromagnetic lens. The annular
gap and pole piece increase the intensity of the magnetic field. The electrons follow a spiral
path through the lens. (Original.)
Furthermore, changes in magnification in the electron microscope require only a
change in the focal length of the projector lens; the magnification of the objective lens is
fixed. Finally, the electromagnetic lenses have a considerable depth of field, so that fine
changes in focus are not readily appreciable by direct visual observation; accurate focusing
must, therefore, be carried out by viewing the fluorescent screen with a binocular
microscope.
Xll
Introduction to Electron Microscopy
A further difference between light and electron microscopy is in image formation. In
the former the image is due to differential absorption of light, which can be accentuated
by staining, and results in visible differences in intensity and wavelength between parts of
the image (contrast). In the electron microscope absorption of the electrons by a
specimen of useful thickness is small. Electrons giving up their energy to the specimen are
absorbed, electrons giving up part of their energy are inelastically scattered, and those
giving up no energy to the specimen, but which change direction, are elastically scattered.
The electron image is due to the scattering of electrons by their collision with electrons
and atoms in the specimen. Those electrons which are strongly scattered are unable to
pass through the lens aperture and are lost. This loss of electrons at a particular point of
Fluorescent screen
vA- Eye I
-Final image"
Projector lens
*~\ ' ' / 7
□\ /
\ /
\ /
\//rwn
Objective lens
Specimen—
Condenser lens
\ /
\ /
\/
v_
Source Γ01
/ \
Light Electrons
FIG. 1.2. A diagram showing the similarities and differences of light and electron micro
scopes. (Original.)
the specimen results in reduced intensity at that point on the fluorescent screen, i.e. it
appears dark or electron-dense. The amount of scatter by any area or structure in the
specimen depends on its physical density and thickness (mass density) and not on the
atomic number of the atoms composing that structure, or its chemical composition. By
the addition of heavy metals or other substances to a particular structure, the scattering
power of that structure can be increased and, therefore, appear more electron-dense on
the screen. Such techniques are called electron staining and increase the contrast between
the electron-dense and electron-lucid parts of the specimen, so aiding discrimination and
interpretation of the image. Contrast in the image, therefore, is due to the accumulative
effects of electron scattering which is visualized as variations in intensity over the viewing
screen, photographic plate, or photographic print.
Xlll
Introduction to Electron Microscopy
Specimen Preparation
The electron microscope when allied with a particular technique or techniques can be
used to investigate a variety of structural parameters of a biological specimen. It is often
necessary, however, for the investigator to select a single parameter and tailor his
technique to suit, for example he may wish to see the surface topography, in which case
the internal structure may be obscured, or he may desire to study in detail internal
features, thereby perhaps denying himself information about the tridimensional configura
tion of the specimen.
The preparative techniques of electron microscopy are many and varied but may be
classified for convenience into those designed for examination of a whole specimen, be it
a complete cell or only a fragment of a single organoid, and those designed for
examination of thin sections of the specimen.
1. Whole Specimen Techniques
(a) DIRECT TRANSMISSION
The specimen to be examined is placed in the microscope with little or no
pretreatment and must, therefore, be virtually dry and resistant to distortion by heat. If
thin, the specimen may be featureless, due to the inherent lack of electron scattering of
biological material, or show poorly defined internal features. If thick, only a silhouette
will appear, since all electrons which strike the specimen will be scattered or adsorbed
(Fig. 1.3a). Generally no surface topography is visible, evidence of internal structure is
poor, and structures appear only in silhouette.
(b) SHADOW TECHNIQUE
This technique increases the electron density of the specimen in such a way as to
provide some evidence of its surface features. By heating a small piece of metal in vacuo
(gold/palladium or platinum) a metal film is deposited at an angle to the specimen,
which lies on a supporting film. Areas of the specimen shielded from the stream of metal
particles because of the angle of deposition are uncoated and therefore electron
transparent, so appearing "light". Areas coated with metal scatter electrons and appear
"dark" when viewed in the microscope. In the electron micrographs, the image is usually
reversed so that the "light" regions appear dark, like shadows, and the metal-coated areas,
therefore, appear as lucid regions. From the known angle of metal deposition and the
length of the shadow, the height of the specimen at a particular point may be calculated
and the shadow gives an accentuated outline of the surface topography (Figs. I.3b, I.4a).
(c) REPLICAS
If the specimen is thick or heat labile, the surface detail can be revealed by
replication, that is by the making of an accurate copy in a more electron transparent and
stable material such as carbon or plastic. Making a replica may involve a number of stages
and be quite a complex procedure. The replica once formed, however, is either stripped
off the specimen or the specimen is digested away (Figs. I.4b, 1.5a, b, c.) Replicas are
often shadow cast so as to increase the three-dimensional appearance of the image.
(d) NEGATIVE STAINING
This technique is capable of revealing both the outline and the internal structure of
the specimen, since the stain can penetrate between and around its substructures or
molecules.
XIV
