- Index
A . Parents´
house, family
- B. School,
vocational choice
- C. The
cathode-ray oscillograph and the short coil
- D. Why
I pursued the magnetic electron lens for the electron microscope
- E. The
invention of the electron microscope
- F. How
the industrial production of electron microscopes came to be
- G. Development
of electron microscopy after 1945
A . Parents´ house, family
A month ago, the Nobel Foundation sent me its yearbook of 1985. From
it I learnt that many Nobel lectures are downright scicntific lectures,
interspersed with curves, synoptic tables and quotations. I am somewhat
reluctant to give here such a lecture on something that can be looked up
in any modern schoolbook on physics. I will therefore not so much report
here on physical and technical details and their connections but rather
on the human experiences - some joyful events and many disappointments
which had not been spared me and my colleagues on our way to the final
breakthrough. This is not meant to be a complaint though; I rather feel
that such experiences of scientists in quest of new approaches are
absolutely understandable, or even normal.
In such a representation I must, of course, consider the influence of
my environment, in particular of my family. There have already been some
scientists in my family: My father, Julius Ruska, was a
historian of sciences in Heidelberg and Berlin; my uncle, Max Wolf,
astronomer in Heidelberg; his assistant, a former pupil of my father and
my godfather, August Kopff, Director of the
Institute for astronomical calculation of the former Friedrich-Wilhelm-University
in Berlin*. A cousin of my mother, Alfred
Hoche, was Professor for Psychiatry in Freiburg/Breisgau; my
grandfather from my mothes side, Adalbert Merx, theologian
in Giessen and Heidelberg.
My parents lived in Heidelberg and had seven children. I was the
fifth, my brother Helmut the sixth. To him I had particularly
close and friendly relations as long as I can rememer. Early, optical
instruments made a strong impression on us. Several times Uncle Max had
shown us the telescopes at the observatory on the Koenigstuhl near
Heidelberg headed by him. With the light microscope as well we soon had
impressive, yet contradictory, relations. In the second floor of our
house, my father had two study rooms connected by a broad sliding door
which usually was open. One room he used for his scientific historical
studies relating to classical philology, the other for his scientific
interests, in particular mineralogy, botany and zoology. When our games
with neighbours kids in front of the house became too noisy, he would
knock at the window panes. Thisusually only having a
brief effect, he soon knocked a second time, this time considerably
louder. At the third knock, Helmut and I had to come to his room and sit
still on a low wooden stool, dos à dos, up to one hour at 2 m distance
from his desk. While doing so we would see on a table in the other room
the pretty yellowish wooden box that housed my father´s big Zeiss microscope,
which we were strictly forbidden to touch. He sometimes demonstrated to
us interesting objects under the microscope, it is true; for good
reasons, however, he feared that childrens hands would damage the
objective or the specimen by clumsy manipulation of the coarse and fine
drive. Thus, our first relation to the value of microscopy was not
solely positive.
B. School, vocational choice
Much more positive was, several years later, the excellent biology
instruction my brother had through his teacher Adolf
Leiber and the very thorough teaching I received through my
teacher Karl Reinig. To my great pleasure I
recently read an impressive report on Reinig´s personality in the
Memoirs of a two-years-older student at my school, the later theoretical
physicist Walter Elsasser. Even today I
remember the profound impression Reinig´s comments made upon me when he
explained that the movement of electrons in an electrostatic field
followed the same laws as the movement of inert mass in gravitational
fields. He even tried to explain to us the limitation of microscopical
resolution due to the wavelength of light. I certainly did not clearly
understand all this then, because soon after that on one of our many
walks through the woods around Heidelberg I had a long discussion on
that subject with my brother Helmut, who already showed an inclination
to medicine, and my classmate Karl Deissler,
who later studied medicine as well.
In our College (Humanistisches Gymnasium), we had up to 17 hours of
Latin, Greek and French per week. In contrast to my father, who was
extremely gifted for languages, I produced only very poor results in
this field. My father, at that time teacher at the same school, daily
learnt about my minus efforts from his colleagues and blamed me for
being too lazy, so that I had some sorrowful school years. My Greek
teacher, a fellow student of my father, had a more realistic view of
things: He gave me for my confirmation the book "Hinter Pflug und
Schraubstock" (Behind plow and vise) by the Swabian poet engineer Max Eyth (1836-1906). I had always been
fascinated by technical progress; in particular I was later interested
in the development of aeronautics, the construction of airships and air
planes. The impressive book of Max Eyth definitely prompted me to study
engineering. My father, having studied sciences at the universities of
Strassburg, Berlin and Heidelberg, obviously regarded study at a
Technical High School as not being adequate and offered me one physics
semester at a university. I had, however, the strong feeling that
engineering was more to my liking and refused.
C. The cathode-ray oscillograph and the short
coil
After I had studied two years electrotechnical engineering in Munich,
my father received a call to become head of a newly founded Institute for theHistory of Sciences in Berlin** in 1927. Thus,
after my pre-examination in Munich I came to Berlin for the second half
of my studies. Here I specialized in high-voltage techniques and
electrical plants and heard, among others, the lectures of Professor
Adolf Matthias. At the end of the summer term
in 1928 he told us about his plan of setting up a small group of people
to develop from the Braun tube an efficient cathode-ray oscillograph for
the measurement of very fast electrical processes in power stations and
on open-air high-voltage transmission lines. Perhaps with the memory of
my physics school lesson in the back of my head, I immediately
volunteered for this task and became the youngest collaborator of the
group, which was headed by Dr. Ing. Max Knoll. My first attempts with experimental
work had been made in the practical physics course at the Technical High
School in Munich under Professor Jonathan
Zenneck, and now in the group of Max Knoll. As a newcomer I was
first entrusted with some vacuum-technical problems which were important
to all of us. Through the personality of Max Knoll, there was a
companionable relationship in the group, and at our communal afternoon
coffee with him the scientific day-to-day-problems of each member of the
group were openly discussed. As I did not dislike calculations, and our
common aim was the development of cathode-ray oscillographs for a
desired measuring capability, I wanted to devise a suitable method of
dimensioning such cathode-ray oscillographs in my Studienarbeit - a prerequisite for being
allowed to proceed to the Diploma examination.
The most important parameters for accuracy of measurement and writing
speed af cathode-ray oscillographs are the diameter of the writing spot
and its energy density. To produce small and bright writing spots, the
electron beams emerging divergently from the cathode had to be
concentrated in a small writing spot on the fluorescent screen of the
cathode-ray oscillograph. For this, already Rankin in 1905 [1]
used a short dc-fed coil, as had been used by earlier experimentalists
with electron beams (formerly called glow or cathode rays ). Even before
that, Hittorf (1869) [2]
and Birkeland (1896) used the rotationally
symmetric field lying in front of a cylindrical magnet pole for
focussing cathode rays. A more precise idea of the effect of the axially
symmetric, i. e. inhomogeneous magnet field of such poles or coils on
the electron bundle alongside of their axes had long been unclear.
Therefore, Hans Busch [3]
at Jena calculated the electron trajectories in
such an electron ray bundle and found that the magnetic field of the
short coil has the same effect on the electron bundle as has the convex
glass lens with a defined focal length on a light bundle. The focal
length of this magnetic electron lens can be changed continuously by
means of the coil current. Busch wanted to check experimentally his
theory but for reasons of time he could not carry out new experirnents.
He made use of the experimental results he had already obtained sixteen
years previously in Goettingen. These were, however, in extremely
unsatisfactory agreement with the theory. Perhaps this was the reason
that Busch did not draw at least the practical conclusion from his lens
theory to image some object with such a coil.
In order to account more precisely for the properties
of the writing spot of a cathode-ray oscillograph produced by the short
coil, I checked Busch´s lens theory with a simple experimental
arrangement under better, yet still inadequate, experimental conditions
(Fig.
1) and thereby found a better but still not entirely satisfactory
agreement of the imaging scale with Busch´s theoretical
 |
expectation. The main reason was that I had
used a coil of the dimensions of Busch s coil whose field
distribution along the axis was much too wide. My Studienarbeit [4],
submitted to the Faculty for Electrotechnical Engineering in 1929,
contained numerous sharp images with different magnifications of
an electron-irradiated anode aperture of 0.3 mm diameter which had
been taken by means of the short coil (magnetic electron lens ) -
i. e. the first recorded |
|
Fig. 1: Sketch by the author (1929) of the
cathode ray tube for testing the imaging properties of the
non-uniform magnetic field of a short coil [4, 5]. |
electron-optical images.
Busch´s equation for the focal length of the magnetic field of
a short coil implied that a desired focal length could be produced
by the fewer Ampere turns the |
more the coil
field was limited to a short region alongside the axis, because in that
case the field maximum is increased. It was therefore logical for me as
a prospective electrotechnical engineer to suitably envelop the coil
with an iron coating, with a ring-shaped gap in the inner tube.
Measurements at such a coil immediately showed that the same focal
length had been reached with markedly fewer Ampere turns [4,
5].
Vice versa, in this manner a shorter focal length can, of course, also
be obtained by an equal number of Ampere turns.
D. Why I pursued the magnetic electron lens for the
electron microscope
In my Diploma Thesis (1930) I was to search for an
electrostatic replacement for the magnetic concentration of the
divergent electron ray bundle, which would probably be easier and
cheaper. To this end, Knoll suggested experimental investigation of an
arrangement of hole electrodes with different electrical potential for
which he had taken out a patent a year before [6].
We discussed the shape of the electric field between these electrodes,
and I suggested that because of the mirror-like symmetry of the
electrostatic field of the electrodes on either sidc of the lens centre,
a concentrating effect of the curved equipotential planes in the hole
area could not take place. I only had the field geometry in mind then.
But this conclusion was wrong. I overlooked that as a consequence of the
considerably varying electron velocity on passage through such a field
arrangement, a concentration of the divergent electron bundle must, in
fact, occur. Knoll did not notice this error either. Therefore I pursued
another approach in my Diploma Thesis [7].
I made the electron bundle pass a bored-out spherical condenser with
fine-meshed spherically shaped grids fixed over each end of the bore.
With this arrangement I obtained laterally inverted images in the
correct imaging scale. Somewhat later I found a solution which was
unfortunately only theoretically correct. In analogy to the refraction
of the light rays on their passage through the optical lens at their
surfaces ("Grenzflaechen"), I wanted to use, for the electrical lens,
the potential steps at corresponding surfaces, which are shaped like
glasses lenses [8].
Thus, the energy of the electron beams is temporarily changed - just
like light beams on passage through optical lenses. For the realization
of this idea, on each side of the lens two closely neighboured
fine-meshed grids of the shape of optical lenses are required which must
be kept on electrical potentials different from each other. First
attempts confirmed the rightness of this idea, but at the same time also
the practical inaptness of such grid lenses because of the too strong
absorption ofthe electron beam at the four grids and due
to the field distribution by the wires.
As a consequence of my false reasoning and the experimental
disappointment I decided to continue with the magnetic lens. I only
report this in so much detail to show that occasionally it can be more a
matter of luck than of superior intellectual vigor to find a better - or
perhaps the only acceptable way. The approach of the transmission
electron microscope with electron lenses of electrostatic hole
electrodes was later pursued by outstanding experimentalists in other
places and led to considerable initial success. It had, however, to be
abandoned because the electrostatic lens was for physical reasons
inferior to the magnetic electron lens.
E. The invention of the electron microscope
After obtaining my Degree (early 1931), the economic situation had
become very difficult in Germany and it seemed not possible to find a
satisfactory position at a University or in industry. Therefore I was
glad that I could at least continue my unpaid position as doctorand in
the high-voltage institute. After having shown in my Studienarbeit of
1929 that sharp and magnified images of electron-irradiated hole
apertures could be obtained with the short coil, I was now interested in
finding out if such images - as in light optics - could be further
magnified by arranging a second imaging stage behind the first stage.
Such an apparatus with two short coils was easily put together (Fig.
2) and in April 1931 I obtained the definite proof that it was
possible (Fig.
3). This apparatus is justifiably regarded today as the first
electron microscope even though its total magnification of 3.6 x 4.8 = 14.4 was extremely modest.
 |
 |
|
Fig. 2: Sketch by the author (Mar 9, 1931)
of the cathode raytube for testing one-stage and two-stage
electron-optical imaging by means of two magnetic electron lenses
(electron microscope) [8]. |
- Fig. 3: First experimental proof (Apr
7, 1931) that speciemens (aperture grids) irradiated by
electrons can be imaged in magnidied form not only in one but
also in more than one stage by means of (magnetic) electron
lenses. (U=50 kV) [8].
- a) one-stage image of the platinum grid in
front of coil 1 by coil 1; M=13X
- b) one-stage image of the bronze grid in front
of coil 2 by coil 2; M=4.8X
- c) two-stage image of the platinum grid in
front of coil 1 by coil 1 and coil 2; M=17.4X together with the
one -stage image of the bronze-grid in front of coil 2 by coil
2; M=4.8X
- kk - cold cathode; Pt N - platinum grid; Sp 1
- coil 1; Br N - bronze grid; Sp 2 - coil 2; LS - fluorescent
screen.
|
The first proof had thus been given that - apart from light and glass
lenses - images of irradiated specimens could be obtained also by
electron beams and magnetic fields, and this in even more than one
imaging stage. But what was the use of such images if even grids of
platinum or molybdenum were burnt to cinders at the irradiation level
needed for a magnification of only 17.4 x. Not wishing to be accused of
showmanship, Max Knoll and I agreed to avoid the term electron
microscope in the lecture Knoll gave in June 1931 on the progress in the
construction of cathode ray oscillographs where he also, for the first
time, described in detail my electron-optical investigations [9,
10].
But, of course, our thoughts were circling around a more efficient
microscope. The resolution limit of the light microscope due to the
length of the light wave which had been recognized 50 years before by Ernst Abbe and others could, because of lack of
light, not be important at such magnifications. Knoll and I simply hoped
for extremely low dimensions of the electrons. As engineers we did not
know yet the thesis of the "material wave" of the French physicist de Broglie [11]
that had been put forward several years earlier (1925). Even physicists
only reluctantly accepted this new thesis. When I first heard of it in
summer 1931, I was very much disappointed that now even at the electron
microscope the resolution should be limited again by a wavelength (of
the "Materiestrahlung"). I was immediately heartened, though, when with
the aid of the de Broglie equation I became satisfied that these waves
must be around five orders ofmagnitude shorter in length
than light waves. Thus, there was no reason to abandon the aim of
electron microscopy surpassing the resolution of light microscopy.
In 1932 Knoll and I dared to make a prognosis of the
resolution limit of the electron microscope [12].
Assuming that the equation for the resolution limit of the light
microscope is valid also for the material wave of the electrons, we
replaced the wave length of the light by the wave length of electrons at
an accelerating voltage of 75 kV and inserted into the Abbe relation the
imaging aperture of 2 x10-2 rad which is what we had used
previously. This imaging aperture is still used today. Thereby, that
early we came up with a resolution limit of 2.2 Å = 2.2 x
10-1m, a value that was in fact obtained 40 years later.
Of course, at that time our approach was not taken seriously by most
of the experts. They rather regarded it as a pipe-dream. I myself felt
that it would be very hard to overcome the efforts still needed - mainly
the problem of specimen heating. In April 1932, M. Knoll had taken up a
position with Telefunken (Berlin) involving developmental work in the
field of television.
In contrast to many biologists and medical scientists, my brother Helmut,
who had almost completed his medical studies, believed in considerable
progress for these disciplines should we be successful. With his
confidence in a successful outcome he encouraged me to overcome the
expected difficulties. In a next step I had to show that it was possible
to obtain sufficiently high magnifications to prove a
better-than-lightmicroscope resolution. To this effect a coil shape had
to be developed whose magnetic field was compressed to a length that
small of the coil axis to allow short focal lengths as are needed forhighly magnified images in not too great a distance behind
the coil. The technical solution for this I had already given in my
Studienarbeit of 1929 with the iron-clad coil. In 1932 I applied -
together with my friend and co-doctorand Bodo v. Borries -
for a patent on the optimization of this solution [13],
the "Polschuhlinse", which is used in all magnetic electron microscopes
today. Its realization and the measuring of the focal lengths which
could be verified with it were subject of my thesis [14].
It was completed in August 1933, and in my measurements I obtained focal
lengths of 3 mm for electron rays of 75 kV acceleration (Fig.
4).
 |
 |
|
Fig. 4: Cross-section of the first
polepiece lens [14, 15] |
Fig. 5: First (two-stage) electron
microscope magnifying higher than the light microscope.
Cross-section of the microscope column (re-drawn 1976) [15]. |
Of course, now with these lenses I immediately wanted to design a
second electron microscope with much higher resolving power. To carry
out this task I obtained by the good offices of Max von Laue for the second halfyear of 1933 a
stipend of Reichsmark 100 per month from the "Notgemeinschaft der
Deutschen Wissenschaft" to defray running costs and personal expenses.
Since I had completed the new instrument by the end of November (Fig.
5), I felt I ought to return my payment for December. To my great
joy, however, I was allowed to keep the money as an exception.
Nevertheless, this certainly was the cheapest electron microscope ever
paid for by a German organization for the promotion of science.
For reasons explained in the beginning of the next chapter, I
accepted a position in industry on December 1, 1933. Therefore I could
only make a few images with this instrument which magnified 12000 x [15],
but I noticed a decisive fact which gave me hope for the future: Even
very thin specimens yielded sufficient contrast, yet no longer by
absorption but solely by diffraction of the electrons, whereby - as is
known - the specimens are heated up considerably less.
F. How the industrial production of
electron microscopes came to be
I also realized, however, that the further developmeni of a
practically useful instrument with better resolution would require a
longer period of time and enormous costs. In view of the results
achieved there was little hope of obtaining financial support from any
side for the time being. I was prepared for a longer dry spell and
decided to approach the goal of a commercial instrument later, together
with Bodo v. Borries and my brother Helmut. Therefore, Iaccepted a position with the "Fernseh AG" in
Berlin-Zehlendorf where I was engaged in the development of Braun tubes
for image pick-up and display tubes. In order to better coordinate our
efforts to obtain financial support for the production of commercial
electron microscopes, I convinced Bodo v. Borries to give up his
position at the "Rheinisch-Westfaelische Elektrizitaetswerke"
at Essen and return to Berlin. Here, he found a
position at "Siemens-Schuckert" in 1934. We
approached many governmental and industrial research facilities for
financial help.
During this period, first electron micrographs appeared of biological
specimens. Heinz Otto Mueller (student in
electrotechnical engineering) and Friedrich
Krause (medical student) worked at the instrument I had built in
1933, and they published increasingly better results (Figs. 6 to 9).
|
|
 |
 |
|
- Fig. 6: Wing surface of the house fly.
(First internal photography; U=60 kV,
Mel=2200)
- (Driest, E. and Mueller, H. O.: Z.Wiss.
Mikroskopie 52, 53-57 [1935])
|
- Fig. 7: Diatoms "Amphipleura
pellucida".
- (U=53 kV, Mel =3500;
¶"=130nm)
- (F. Krause in: Busch, H., and Brueche, E.:
Beitraege zur Elektronenoptik, 55-61, Verl. Hoh. Ambrosius
Barth, Leipzig 1937)
|
- Fig. 8: Bacteria (culture infusion),
fixed with formalin and embedded in a supporting fiml stained
with heavy metal salt.
- (U=73.5 kV, Mel =2000)
- (Krause, F.: Naturwissenschaften 25, 817-825
[1937])
|
- Fig. 9: Iron Whisker
- (U=79 kV, Mel =3100)
(Beischer, D. and Krause, F.: Naturwissenschaften
25, 825-829 [1937]) |
Unfortunately these two very gifted young scientists did not survive
the II. World War.
At Brussels Ladislaus Marton had built his first horizontal
microscope and obtained relatively low magnifications of biological
specimens [17].
In 1936 he built a second instrument, this time with a vertical column
[18].
In spite of these more recent publications, it took
us three years to be successful in our quest for financial support
through the professional assessment of Helmut Ruska´s former clinical
teacher, Professor Dr. Richard Siebeck, Director of the I. Medical
Clinic of the Berlin Charité. I quote two
paragraphs of his assessment of October 2, 1936 [19]:
"If these things were to be realised it hardly needs to be
emphasised that the advances in the field of research into the causes
of disease would be of immediate practical interest to the doctor. It
would deeply affect real problems concerned to a large extent with
diseases of growing clinical significance and thus of great importance
for public health.
Should the possibilities of microscopical resolution exceed thc
assumed values by a factor of a hundred, the scientific consequences
would be incalculable. What seems attainable now, I consider to be so
important, and success seems to me so close, that I am ready and
willing to advise on medical research work and to collaborate by
making available the resources of my Institute".
This expertise impressed Siemens in Berlin and Carl Zeiss in Jena***, and they were both ready
to further the development of industrial electron microscopes. We
suggested the setting up of a common development facility in order to
make use of the electrotechnical expertise of Siemens and the know-how
in precision engineering of Zeiss, but unfortunately the suggestion was
refused and so we decided in favour of Siemens. As first collaborators
we secured Heinz Otto Mueller for the practical development and Walter Glaser from Prag as theorist. We started
in 1937, and in 1938 we had completed two prototypes with condenser and
polepieces for objective and projective as well as airlocks for
specimens and photoplates. The maximum magnification was 30.000 x [20].
One of these instruments was immediately used for first biological
investigations by Helmut Ruska and several medical collaborators. (H.
Ruska was released from Professor Siebeck for our work at Siemens.)
Unfortunately, for reasons of time I cannot give here a survey of this
fruitful publication period.
In 1940, upon our proposal Siemens set up a guest
laboratory, headed by Helmut Ruska, with four electron microscopes for
visiting scientists. Helmut Ruska could show first images of
bacteriophages in 1940. An image taken somewhat later (Fig.
10)
 |
 |
- Fig. 10: Bacteriophages.
- (Ruska, H.: Naturwissenschaften 29, 367-368
(1941) and Arch. Ges. Virusforsch. 2, 345-387 (1942).)
|
Fig. 11: The first serially produced
electron microscope by Siemens. General view [21]. |
clearly shows the shape of these tiny hostile bacteria. This
laboratory was destroyed during an air raid in the autumn of 1944.
Very gradually now interest in electron microscopy was growing. A
first sales success for Siemens has been achieved in 1938 when the
chemical industry which was represented largely by "IG Farbenindustrie"
placed orders for an instrument in each of their works in Hoechst,
Leverkusen, Bitterfeld and Wolfen. The instrument was only planned at
the time, however not yet built or even tested. By the end of 1939 the
first serially produced Siemens instrument [21]
had been delivered to Hoechst (Fig.
11). The instrument No. 26 was, by the way, delivered to Professor
Arne Tiselius in Uppsala in autumn 1943. By
Februrary 1945 more than 30 electron microscopes had been built in
Berlin and delivered. Thus, now also independent representatives of
various medical and biological disciplines could form their own opinions
about the future prospects of electron microscopy. The choice of
specimens was still limited though, since sufficiently thin sections
were not yet available. The end of the war terminated the close
cooperation with my brother and B. v. Borries.
G. Development of electron
microscopy after 1945
Our laboratory had to be reconstructed completely. I could start
working with mainly new coworkers as early as June 1945. In spite of
difficult conditions in Berlin and Germany, newly developed electron
microscopes [22]
could be delivered by the end of 1949. In 1954 Siemens had regained its
former leading position with the "Elmiskop" [23]
(Fig.
12 and Fig.
13).
 |
 |
|
Fig. 12: The first serially produced 100
kV-electron microscope with two condenser lenses for "small region
radiation" by Siemens (cross-section) [23]. |
Fig. 13: Same instrument as in Fig. 12
(general view) [23]. |
This instrument had, for the first time, two condenser lenses
allowing thermal protection of the specimen by irradiating only the
small region that is required for the desired final magnification. Since
now, for a final magnification of 100.000 x a specimen field of only 1
µm must be irradiated for an image of 10 cm diameter in contrast to
earlier irradiation areas of about 1 mm diameter, the power of the
electron beam converted into heat in the object can be reduced down to
the millionth part. The specimens are heated up just to the extent that
the heat power produced can be radiated into the entire region around
the object. If the heat power is low, a lower temperature rise with
respect to the environment results.
The new instrument was, however, a big disappointment at first when
we realized that at this "small region radiation" the image of the
specimen fields, which was now no longer hot, became so dark within
seconds that all initially visible details disappeared. Investigations
then showed that minor residual gases in the evacuated instrument,
particularly hydrocarbons, condensed on the cold inner planes of the
instrument, i.e. they now even condensed on the specimen itself. The
image of the resulting C layer in the irradiated specimen field becomes
darker with increasing thickness of the layer. Happily, also this hurdle
could, after some time, be surmounted by relatively simple means: The
entire environment of the specimen was cooled by liquid air so that the
specimen was still markedly warmer than its environment, even without
being heated up by the beam. Thus, the residual gases of hydrocarbons
condensed on the low-cooled planes and no longer on the specimen.
Along with the successful solution of this problem, another
difficulty, that of specimen thickness, had also surprisingly been
overcome by newly developed "ultramicrotomes". Instead of the ground
steel knives whose blades were not sufficiently smooth due to
crystallization, glass fracture edges were used which had no crystalline
unevenness. The usual mechanical translation of the material
perpendicular to the knife is - because of mechanical backlash or even
oil layers - not sufficiently precise for the desired very small
displacements of ~10-5 mm. Smallest displacements free of
flaws were obtained by thermal extension of a rod at whose ends the
specimen to be cut was fastened. In order to keep the extremely thin
sections smooth, they were dropped into an alcoholic solution
immediately after being cut so that they remained entirely flat.
Moreover, more suitable fixing agents had been found for the new cutting
techniques. The development of these new ultramicrotomes considerably
reduced the limitation in the choise of specimens for electron
microscopy. For 25 years now, almost all disciplines furthered by light
microscopy have also been able to benefit from electron microscopy.
During the last decades, electron microscopy has been advanced in
manycountries by numerous leading scientists and
engineers through new ideas and procedures. I can here only give a few
examples: Fig. 14 shows a cross-section through an electron microscope
with single-field condenser objective, the specimen being in the field
maximum of a magnetic polepiece lens [24].
 |
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|
Fig. 14: cross-section through an electron microscope with
single-field condenser objective, the specimen being in the field
maximum of a magnetic polepiece lens [24]. |
Fig. 15: Same instrument as in Fig. 14
(general view) [24]. |
Thereby, the region of increasing magnetic field in front of the
specimen behaves like a condenser of short focal length and the
decreasing field region behind thespecimen as an
objective of equal focal length. With this arrangement both lenses have
a particularly small spherical aberration. Fig. 15 gives a view of the
same instrument. Fig. 16 shows an image obtained with this instrument of
a platelet of a gold crystal. One can clearly see lattice planes
separated by a distance of 1.4 Å. Two such instruments have been further
developed in the Institute for Electron Microscopy, which had been set
up for me in 1957 by the Max-Planck-Gesellschaft
after I had left Siemens. Fig.
17 shows a 3 MV highvoltage instrument developed by Japan Electron Optics Laboratory Co. Ltd. With such
instruments whose development was mainly promoted by Gaston Dupouy (1900-1985),
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|
Fig. 16: plate-like gold crystal, lattice
planes with a separation of 0.14 nm, taken with axial
illumination.
(U=100 kV, Mel=800.000); taken (1976) by K. Weiss
and F. Zemlin with the 100 kV transmission electron microscope
with single-field condenser objective at the Fritz-Haber-Institut of the
Max-Planck-Gesellschaft. |
Fig. 17: 1 MV electron microscope (Japan
Electron Optics Laboratoty Co.
Ltd.). |
apart from extremely high costs, special problems occur in the
stabilization of the acceleration voltage and with the protection of the
operators against X rays. The aim of the development of these
instruments was the investigation of thicker specimens, but now that the
problem of stabilizing the high voltages has been overcome, also the
resolution has been improved by the shorter material wave length of
particularly highly accelerated electrons, so that thinner specimens can
also be investigated.
For quite some time now, the cryotechnique - put forward mainly by Fernandez-Moran in the USA - has been of
increasing importance. With this technique specimens cooled down to very
low temperatures can be studied, because they are more resistant to
higher electron doses, i.e. the mobility inside the specimen is very
much reduced compared to room temperature. Thus, even after unavoidable
ionization, the molecules keep their structure for a long time. In the
last years it has been possible to image very beam-sensitive crystals in
a cryomicroscope with a resolution of 3.5 Å [25,
26]
(Fig.
18) [27].
Fig. 18: Paraffin crystal (left: image
taken with minimum dose, right: superposition of 400 subregions of
the left image by menas of the computer) [25]. |
The specimens were cooled down to -269°C.
Direct imaging with sufficient contrast is not possible because
the specimen is destroyed at the beam dose needed for normal
exposure. Therefore, many very lowdose images are recorded and
averaged. Such a single image is very noisy but still contains
sufficient periodical information. The evaluation procedure is the
following: First, the microgram is digitized using the
densitometer so that each image point is given a number which
describes the optical density. The underexposed image of the whole
crystal is divided like a checkerboard by the computer and then a
large number - in our case 400 - of these image sub-regions is
cross-correlated and summed up by the
computer. |
The resulting image corresponds
to a sufficiently exposed micrograph. On the left part in Fig.
18, the initial noisy image of a paraffin crystal is seen; the right
side shows the averaged image. Each white point is the image of a
paraffin molecule. The long paraffin molecules
C44H90 so are vertical to the image plane. With
this procedure electron micrographical images can be processed by the
computer. It is even possible to image threedimensional protein crystals
with very high resolution [27].
The computer is a powerful tool in modern electron microscopy.
I cannot go into detail concering the transmission electron
microscopes with electrostatic lenses, the scanning electron microscopes
which are widely used mainly for the study of surfaces as well as
transparent specimens, the greatimportance of various
image processing methods carried out partly by the computer, the
field-electron microscope and the ion microscope.
The development of the electron microscopy of today was mainly a
battle against the undesired consequences of the same properties of
electron rays which paved the way for sub-light-microscopical
resolution. Thus, for instanee, the short material wavelength -
prerequisite for good resolution - is coupled with the undesired high
electron energy which causes specimen damage. Thedeflectability in the magnetic field, a precondition for
lens imaging, can also limit the resolution if the alternating magnetic
fields in the environment of the microscope are not sufficiently
shielded by the electron microscopy. We should not, therefore, blame
those scientists today who did not believe in electron microscopy at its
beginning. It is a miracle that by now the difficulties have been solved
to an extent that so many scientific disciplines today can reap its
benefits.
- * today: Humboldt-University Berlin
- ** today: Institut für Geschichte der Medizin - Charité
Berlin
