- 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
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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
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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].
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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
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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.
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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].
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- 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.
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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).
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Fig. 4: Cross-section
of the first polepiece lens
[14,
15]
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Fig. 5: First
(two-stage) electron microscope magnifying
higher than the light microscope. Cross-section
of the microscope column (re-drawn 1976)
[15].
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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).
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- 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])
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- 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)
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- 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])
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- Fig. 9: Iron
Whisker
- (U=79 kV, Mel
=3100)
(Beischer, D. and Krause, F.:
Naturwissenschaften 25, 825-829
[1937])
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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)
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- Fig. 10:
Bacteriophages.
- (Ruska, H.:
Naturwissenschaften 29, 367-368 (1941) and
Arch. Ges. Virusforsch. 2, 345-387
(1942).)
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Fig. 11: The first
serially produced electron microscope by
Siemens. General view [21].
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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).
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Fig. 12: The first
serially produced 100 kV-electron microscope
with two condenser lenses for "small region
radiation" by Siemens (cross-section)
[23].
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Fig. 13: Same
instrument as in Fig. 12 (general view)
[23].
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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].
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Fig. 15: Same
instrument as in Fig. 14 (general view)
[24].
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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.
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Fig. 17: 1 MV electron
microscope (Japan Electron Optics Laboratoty Co.
Ltd.).
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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].
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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.
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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
