Table Of ContentAdvances in Structure Research
by Diffraction Methods
Fortschritte der Strukturforschung
mit Beugungsmethoden
Edited by
W. HOPPE and R. MASON
Volume 7
Unconventional Electron Microscopy
for Molecular Structure Determination
W. HOPPE and R. MASON (Eds.)
Unconventional
Electron Microscopy
for Molecular
Structure Determination
With contributions of
B. M. SIEGEL· A. V. CREWE· M. S. ISAACSON· E. ZEITLER
E. KELLENBERGER· J. KISTLER· V. E. COSSLETT
O. SCHERZER· J. GASSMANN· W. HOPPE· D. TYPKE
With 88 figures
Friedr. Vieweg & Sohn Braunschweig/Wiesbaden
CIP-Kurztitelaufnahme der Deutschen Bibliothek
Unconventional electron microscopy for molecular
structure determination/W. Hoppe and R. Mason (eds.)
With contributions of B.M. Siegel ... -
Braunschweig, Wiesbaden: Vieweg, 1979.
(Advances in structure research by diffraction
methods; vol. 7)
ISBN-13: 978-3-528-08117-1 e-ISBN-13: 978-3-322-86362-1
DOl: 10.1007/978-3-322-86362-1
NE: Hoppe, Walter (Hrsg.]; Siegel, Benjamin M.
(Mitarb.]
1979
All rights reserved
© Friedr. Vieweg & Sohn Verlagsgesellschaft mbH, Braunschweig 1979
Softcover reprint of the hardcover 1st edition 1979
No part of this publication may be reproduced, stored in a retrieval system or transmitted
mechanically, by photocopies, recordings or other means, without prior permission of the
copyright holder.
Set by Vieweg, Braunschweig
ISBN-13: 978-3-528-08117-1
Preface
Generally it is not sufficiently appreciated that electron microscopy is in fact a diffraction
method. In essential aspects electron microscopes are more closely related to X-ray diffracto·
meters than to light microscopes. In electron microscopes monochromatized radiation and
coherent illumination (never used in light microscopy) correspond in X-ray diffractometers
to the primary beam with a small divergence. Imaging ina general sense can take place in
interference experiments between a primary beam and a scattered beam, or between diffe
rent deflected scattered beams. This leads to the realization of an old dream in diffracto
metry, namely to a general experimental solution of the "phase problem". The most im
pressive analogy, however, concerns the potential of the electron microscope as a tool for
structure determination (where the radiation wavelenght is smaller than the atomic distan
ces). It was therefore considered timely to treat this topic in this series. It was a fortunate
cioncidence that in 1976 a Workshop on "Unconventional Electron Microscope Methods
for the Investigation of Molecular Structures" (sponsored by the European Molecular
Biology Organisation, the Deutsche Forschungsgemeinschaft and the Max-Planck-Gesell
schaft) took place, and that most speakers presenting introductory lectures agreed to
publish their contributions in an expanded version in this volume. This volume is thus not
a symposium report in the usual sense since it contains the majority of these introductory
lectures only. For completeness an article on scanning transmission electron microscopy and
on electron microscopy of periodical arrays has been added. This book is meant as an intro
duction into a new and fascinating topic.
For more detailed information the reader might find it useful to consult the reports
of the various conferences on electron microscopy. The first symposia dedicated to this
special subject of unconventional electron micrsocopy were held in London and Hirschegg.
The progress reports of the London Symposium were published in Huxley H. E. and
Klug A., New developments in electron microscopy London, The Royal Society (1971).
The contributions to the Hirschegg Symposium are published in Ber. Buns. Ges. phys.
Chemie (1970) from p. 1090-1216.
The Editors
Contents
B.M. Siegel
Instrumentation: Progress and Problems 1
A. V. Crewe, M. S. Isaacson and E. Zeitler
Progress in Scanning Transmission Electron Microscopy at the University
of Chicago. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 23
E. Kellenberger and J. Kistler
The Physics of Specimen Preparation 49
V. E. Cosslett
Radiation Damage: Experimental Work 81
o.
Scherzer
Radiation Damage: The Theoretical Background ....................... 101
J.G~mann
The Electron Microscope, a Diffractometer to Measure Scattering Amplitudes
and Phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
W. Hoppe andD. Typke
Three-Dimensional Reconstruction of Aperiodic Objects in Electron Microscopy .. 137
W.Hoppe
Three-Dimensional Low Dose Reconstruction of Periodical Aggregates . . . . . . . .. 191
Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 221
1
Instrumentation: Progress and Problems
Benjamin M. Siegel
School of Applied and Engineeting Physics, Cornell University, Ithaca, New York 14853 USA
The electron microscopes currently available are designed to transfer information
down to resolutions of 2-3 A or in terms of spatial frequencies, 0.3-0.5 A-I. Our present
problems relate to efforts directed toward instrumentation that will provide a better speci
men environment, on the one hand, and that will extend the contrast transfer function
(CTF) of the instrument to 1 A-lor better, on the other. The instrumental problems and
their importance related to specimen environment in high resolution electron microscopy
will be discussed in the later part of this contribution. Basic optical parameters such as
electron wavelength and angular aperture of the objectives lens can be changed to enhance
the CTF and the necessary means are well known, but their execution is far from routine.
The electron wavelength can be decreased to give greater resolving power by going to higher
accelerating voltages. Spherical and chromatic aberration coefficients can be minimized by
using a system of multipoles and when corrected, larger angular apertures can be used to
obtain better resolution. These are basic instrumental approaches to the problem and are
being tried by different groups [1, 2, 3]. Special imaging modes such as tilt or rotatory
cone illumination, and phase plates have also been tried [4, 5, 6] and involve special pro
blems of instrumentation. In principle, computer or analogue processing of the electron
microscope image also can be used to enhance the information obtained, but this whole
area is a subject that is being considered in another contribution in this volume.
Electron Optical Instrumentation
I would like to review the electron optical and instrumental developments in the electron
microscope that have taken place in the recent past and are currently in progress. The
commercial instruments have now been engineered to incorporate many of these advances
and much of the new development work is carried out in the laboratories of the compa
nies producing electron microscopes. However, some of the most innovative and complex
instrumenation cannot reasonably be incorporated in general, commercial instruments
and it is worthwhile to survey the total field.
Field Emission Systems: A necessary condition for obtaining information out to high
spatial frequencies is a source of high brightness, i. e., a small effective source with high
electron flux and small beam divergence. Such a source provides an illuminating beam
with the necessary high spatial and temporal coherence for bright-field, phase-contrast
2 B.M. Siegel
imaging. Our computer experiments indicate that the coherence to the illuminating beam
will not be a factor in deteriorating the CTF of optical systems designed to transmit spa
tial frequencies to 0.5 A -1 if the beam divergence is';;; 0.5 X 10-3 rad. and the energy
spread in the beam is';;; 0.5 eV [7]. Clearly, a field emission source is required if these
conditions are to be satisfied and the tolerance will become more stringent as we attempt
to achieve higher resolutions. The field emission source is also essential for the scanning
transmission electron microscope (STEM), for only with a source of the brightness avail
able with the field emission gun can a small beam probe be realized with an electron flux
high enough to scan the object area in a reasonable time and provide an adequate signal
level so resolution is limited by the aberrations of the lenses and the electron wavelength
rather than the statistical noise in the electron signal [8].
The field emission gun has become a well developed, reliably functioning electron
beam source that is now commercially available, or soon will be, for the majority of the elec
tron microscopes, whether the conventional transmission electron microscope (CTEM) or
STEM. The effective brightness of the gun is set by the electron optics of the gun and the
investigations on electron optical designs have concentrated on minimizing the spherical and
chromatic aberrations in this system. The lower the aberration coefficients, the higher the
brightness that can be realized in the beam currents that can be obtained within the same
focussed probe diameter if the aberrations of the gun can be reduced significantly. All of
the guns operating today use electrostatic lenses to produce and accelerate the beam and
the beam currents available with these electrostatic lens systems are fully adequate for
both STEM and CTEM. Certainly, for biological application where radiation damage sets
the limit on incident flux, the present field emission systems are satisfactory.
Much of the electron optical design has been directed toward making the gun illu
minating system more flexible in its application. We are using an einzellens as a pre-accele
rator lens that injects the beam into the accelerator at its nodal point, providing a convenient
flexible system that can be used over a wide range of beam currents and accelerating vol
tages [9,10]. Veneklasen [11] has reviewed the different configurations that can be used
for various modes of operating CTEM and STEM. This type of flexibility is being incor
porated into the field emission systems that are now being produced or being developed
commercially.
There are applications, particularly in analytical electron microscopy on radiation
resistant materials, where the amount of beam current available in a given probe size formed
with an electrostatic field emission gun can be a limiting factor. We have investigated the
performance of a gun that uses a magnetic pre-accelerator lens and have obtained at least
an order of magnitude increase in beam current available within a given probe diameter
[12]. Figure 1 gives plots of the beam current versus probe radius available from various
gun systems; two field emission systems, one using an electrostatic, the other a magnetic
pre-accelerator lens and guns with thermionic sources, one using LaB6, the other a pointed
tungsten emitter. The plots show that the "cross-over radius" between the field emission
source using a magnetic preaccelerator lens and the best thermionic source, LaB6, is now
-3000 A. This increased "cross-over radius" can be an important consideration for appli
cations requiring high current probes. In high resolution microscopy the most likely appli
cations are in the STEM mode of operation, where the electron optics of the condenser
Instrumentation: Progress and Problems 3
..
~
Q.
E 105~------------~~----~-----4~----~
<
I
Z Fig. 1
IJJ
a:
a: Comparison of probe currents
~ as functions of probe radii ob
(,)
tained for different emitters and
IJJ electron optical systems. The
~
a: solid lines represent plots ob
0..106 tained from experimental data
measured at 20kV (12). (j is the
source brightness; Cst and Cs2
are the spherical aberration co
efficients of the pre-accelerator
lens and the accelerator optics,
respectively; 10 is the total emis
sion current from the field emit
ter tips.
IO~O~2~~--~~~~I~O·3--~~~~~~~--~~
PROBE RADIUS (1)
lens system sets the final resolution limit. At the very least, faster scan rates could be
used but the most important applications that could use higher beam currents would be
in analytical microscopy on specimens that can tolerate these high dosages. The higher
beam currents may allow the use of an energy ftlter or spectrometer in the illuminating
system to provide a beam with a very narrow energy spread. One important result would
be the increased energy resolution that could give enhanced resolution of the fme struc
ture in energy loss spectra. Actually, this narrow energy spread, high beam current system
would also apply to energy loss investigations using the CTEM mode [13].
Superconducting Lenses: There are some distinct advantages that may be gained in both
resolution and SIN by using superconducting lenses, particularly at higher voltages. Of
course, the important gain in resolution is achieved by going to higher accelerating voltages,
where the resolution improves almost directly proportional to the decrease in wavelength
of the electrons, if the relative instrumental stabilities can be maintained. Going from
100 kV to 500 kV should give at least a two-fold gain in resolution with A going from
.037 A to .014 A, if the aberration coefficients of the objective lens can be kept constant.
But, using conventional lenses in which HOmax is set by the saturation of the ferromagnetic
pole-pieces, CsA ::::: constant and Cs must be scaled up with voltage. Thus, some of this po
tential gain in resolution will not be realized. However, by using special configurations of
4 B.M. Siegel
superconducting materials which can produce higher HOrnax and still confme the magnetic
fields to the same half-width, Cs and Cc can be kept constant [14]. The limits here are set
by the available superconducting materials and some of the materials that have become
available since our early experiments indicate very interesting potential improvement. The
gain in resolution should not be overemphasized. The resolution is proportional to C!,4,
so in going from 100 kV to 500 kV with a conventional lens, Cs increases by a factor of
two results only in a gain of about 20 % in the resolution over that which could be obtai
ned with conventional lens.
More important than the gain in resolution is the gain in the image signal to noise or
contrast that can be realized in CTEM by keeping Cs constant with decreasing wavelength.
The collection "efficiency" and hence the signal to noise in the imaging system varies as
'fret?, the solid angle of the effective aperture. If the objective lens is defocussed the optimal
amount, that is one wavelength of defocus balancing one wavelength of spherical aberra
tion, the square of the optimum angular aperture is given by o? = 2 VA/Cs and thus the
fraction of the total scattering collected within the aperture is inversely proportional to
C~/2 at a given voltage or wavelength. By using superconducting lenses with an optimum
Cs at higher voltages, there is a substantial gain in the fraction of the total elastic scattering
from the specimen that can be collected. Since the differential scattering cross-section scales
with a/A = constant, the effect is that the scattering is concentrated more and more in the
forward direction as the voltage is increased and the smaller Cs, the larger is the effective
solid angle that can be used to collect this scattering.
We have used a supercondusting lens configuration that does provide the type of
magnetic field confmement desired. A schematic cross-section is shown in Figure 2 [14].
Annular discs, etched to give rings of Nb Sn, are stacked between the pole-pieces of a
3
ferromagnetic yoke that is used to close the magnetic circuit when the field is trapped in
the discs. Since the peak field is at the mid-point of the stack of discs, the pole-pieces do
not saturate, at least up to the 23 k Gauss reached with our present stack of discs, and the
axial field distribution closely approximates a Gaussian shape without the tails usually asso
ciated with lenses in which the fields are shaped by ferromagnetic pole-pieces. The ferro
magnetic yoke is also used as the magnetic circuit to concentrate the external field produ-
FERROMAGNETIC CONTAINER
Fig. 2
Schematic cross-section of the superconducting
disc lens.
Instrumentation: Progress and Problems 5
ced by the excitation coil that is needed to induce the superconducting current in the discs.
The discs are kept above the critical temperature of the Nb Sn by the heater until the
3
desired field is reached. The heater is then switched off and a superconducting current is
produced in the rings of the annular discs. The excitation current is slowly turned down
and the superconducting current remains trapped in the discs. This persistent mode of
operation produces a very stable magnetic field. With the material we had available, trapped
peak fields up to 23 k Gauss were produced with half-widths of 1.76 mm in a lens with a
3 mm bore diameter and 2.5 mm gap. The limit in this case was set by the low packing fac
tor (0.21) of the available Nb Sn discs. With some of the newer material now available and
3
a different configuration for energizing the lens, we expect to reach considerably higher
peak fields with equivalent half-widths.
One of the most interesting and succesful superconducting lens configurations that
has been developed is the shielding lens of Dietrich and co-workers [15]. The basic confi
guration is shown in the schematic in Figure 3. Two co-axial cylinders of very homoge
neous Nb Sn are used to exclude the external magnetic field except in the small gap be
3
tween the cylinders. High magnetic fields are produced by superconducting excitation coils
outside the cylinders and axially symmetric magnetic fields with peak fields as high as 50-
60 k Gauss have been obtained. First order astigmatism caused by nonaxial field distribu
tions are correctable with shim coils in the gap. This configuration requires side loading of
the specimen and aperture, if the lens is to be practical and an objective lens with side load
ing has been operated succesful at high resolution. This lens was designed to operate up to
voltages of 400 kV and has been tested up to 250 kV giving a resolution of better than
2 A (16].
An ingeneous extention of the shielding lens principle has been proposed and is being
developed by Hoppe and co-workers'[ 17]. By inserting a coaxial Nb Sn shielding ring in
3
_ [5]
superconductlng Iron
shielding material
50mm
2
Fig_ 3
:J Schematic cross-section showing
the liquid He cryostat containing
the electron optical lens for a
4 450kV electron microscope (15).
Legend:
1. condenser lens with s.c. coil
2. deflection coils
8 3. shielding s.c. objective lens
4. side entry specimen part
5. stigmator
6. deflection coils
7. intermediate lenses with s.c. coils
