English Version
Source: D H Kruger, P Schneck, H R Gelderblom * The Lancet, Department of medical history - Volume 355, Number 9216 13 May 2000
Citation: The Lancet 2000; 355: 1713-17
Original title: Helmut Ruska and the visualisation of viruses
Department of Virology (Prof D H Kruger MD) and Medical History (Prof P Schneck MD), Charité Medical School, Humboldt University, D-10098 Berlin; and Robert Koch Institute, D-13353 Berlin, Germany (Prof H R Gelderblom, MD)
Correspondence to: Prof D H Kruger (e-mail: detlev.kruger@charite.de)
Content
• The bacterial cell
• Invisible mysterious pathogens
• Visualisation of viruses
• Further use in virus research
• Scientific tradition of the Charité
• References
In 1939 the Archiv für die Gesamte Virusforschung (now Archives of Virology), the first international journal of virology, was founded. In the opening volume (completed in 1940), there was an article on the significance of “ultramicroscopy” in the evaluation of the nature of viruses by a young trainee in internal medicine, Helmut Ruska (1908-73).[1] The paper was written with his brother, Ernst Ruska (1906-88), and his brother-in-law Bodo von Borries (1905-56). Ernst Ruska received the Nobel Prize in 1986 for “his fundamental work in electron optics, and for the design of the first electron microscope”, 13 years after Helmut’s death. Helmut Ruska (figure 1) is little known now, yet he played an integral and important part during the early development of electron microscopy. Through his interest in the specimen preparation and application of the electronmicroscope to problems in bioscience, he became a major driving force in the development of this new instrument into a tool that became essential to the biomedical sciences.
The bacterial cell
A major step in our present understanding of life was the ability to analyse structures and minute organisms too small to be scrutinised by the naked eye. In the 17th century, a successful beginning was made when Antoni van Leeuwenhoeck (1632-1723) visualised living spermatozoa, erythrocytes, and bacteria using a simple type of light microscope invented and made by himself. He built more than 500 microscopes, which were equipped with a single bi-convex lens, generally ground and polished by himself, and used transmitted light. The results of his research were made public in many letters to the Royal Society of London, thus paving the way to a new, previously invisible world that allowed microbes to be recognised as the cause of devastating infectious diseases.[2,3] The progress in imaging raised high expectations, as stated by Robert Hooke in 1665: “by the help of microscopes, there is nothing too small, as to escape our inquiry”.[4] Hooke’s prognosis turned out to be too optimistic, not least because many optical and mechanical problems remained unsolved at that time. Nevertheless, Leeuwenhoeck’s accomplishments are recognised today as one of the fundamental roots of both scientific microbiology and bioscience.
In the following centuries more advanced light microscopes were constructed and used for the investigation of tissues as well as microbes. In painstaking systematic studies in the second half of the 19th century, Robert Koch (1843-1910) proved that specific microbes are the cause of a number of important infections, such as Bacillus anthracis in 1876, Mycobacterium tuberculosis in 1882, and Vibrio cholerae in 1883.[5] Koch’s successful strategy was based on what was later called the Koch-Henle postulate: the regular isolation of the bacterium from infected tissues by single-colony growth on artificial media, the documentation of the specific morphology of the isolate by light microscopy, and the experimental generation of the disease in a suitable animal followed by the final microscopic proof of identity of the bacteria.
Invisible mysterious pathogens
However, despite such rapid progress in visualising microbes, there was growing evidence of the existence of agents too small to be seen by light microscopy. When investigating outbreaks of foot-and-mouth disease in cattle in 1897-98, Friedrich Loeffler (1852-1915) and Paul Frosch (1860-1928) described an agent in the vesicle fluids of diseased animals that could not be cultivated on artificial media or detected by light microscopy. Infectivity, however, passed bacterial-tight filters and caused disease when inoculated into healthy cattle or swine. Even at high dilutions, the filtered lymph induced symptoms. By serial inoculation and reisolation experiments, Loeffler and Frosch convinced themselves that the disease was not caused by some toxic effect but rather by an infectious agent, a virus.[6] The causative agent of a transmissible tobacco-plant disease was described by Dimitri Ivanovski (1864-1920) in 1892, and even more convincingly in 1898 by Martinus Beijerinck (1851-1931), as a filterable infectious agent.[7] Moreover, even bacteria were found to be lysed by submicroscopic agents as described in 1915 by Frederick W Twort (1877-1950) and - perhaps independently of his work - in 1917 by Felix d’Herelle (1873-1949).[8,9]
Thus, a clear distinction between bacteria and other mysterious contagious entities gradually developed in the very late 19th and early 20th century.[10] The emerging concept of the virus was based on the capacity of the agents to pass through filters able to halt bacteria, their invisibility by light microscopy, and their failure to grow on artificial media - ie, outside an infected organism. These negative definitions left open the question of whether these agents represented particulate structures or rather a “contagium fluidum vivum” (Beijerinck), that is, a poorly defined contagious liquid.
The limits of light microscopy are determined by the wavelength of visible light, which is 400-700 nm. For optical reasons, following the famous formula of Ernst Abbe (1840-1905), dmin=l/n sina, the maximum resolving power of the light microscope is limited to about half the wavelength, typically 300 nm. This value is close to the diameter of a rather small bacterium, and viruses cannot therefore be visualised. To attain sublight microscopic resolution, a new type of instrument would be needed, as Ernst Abbe envisaged in 1873.[11] As we know today, accelerated electrons, which have a much smaller wavelength, are used in suitable instruments to scrutinise structures down to the 1 nm range.
Visualisation of viruses
Nearly three centuries had to pass after Leeuwenhoeck’s discoveries, but only 70 years after Abbe’s vision, before viruses were observed directly, thus fostering both the rational understanding of the natural history of a number of infectious diseases and virus research as a basic science. 60 years ago, the first comprehensive presentation of viral structures was published.[1] Helmut Ruska, the first author, was serving as an intern at the Charité Medical School of Berlin University. The publication “Die Bedeutung der Übermikroskopie für die Virusforschung” (”The significance of electron microscopy for virus research”) summarises the intense activities of Helmut Ruska who, in close collaboration with the electrical engineers Ernst Ruska and Bodo von Borries, developed this entirely new field.
Helmut Ruska was born in Heidelberg on June 7, 1908, the sixth of seven children of the historian of science Julius Ruska (1867-1949). He studied medicine from 1927-32 in Berlin, Innsbruck, and Heidelberg, where he graduated as a doctor with a biochemical thesis prepared under the supervision of Ludolf von Krehl (1861-1937). From 1933 to 1940 he worked in several hospitals in Heidelberg and Berlin. In Berlin he increasingly began to focus on the scientific and practical problems of electron microscopy, particularly on its application to biomedical problems.
By the early 1940s, Helmut Ruska had published about 20 reports on submicroscopic structures of bacteria, parasites, and different viruses - eg, poxviruses1,[12] (figure 2), tobacco-mosaic virus,[13] varicella-zoster virus,[14] and bacteriophages (bacterial viruses).[15] He summarised his principal research into the nature and biology of bacteriophages in a thesis and, in 1943, became a lecturer in medicine at Berlin University. During the following decades, bacteriophage and tobacco-mosaic virus became the preferred objects in biophysics and genetics research. Studies on the biology of these particular viruses formed the most important roots of modern virology and molecular genetics.[16,17]
As shown in volume 1 of Archives of Virology (1940).[1] The particles show the brick-shaped outline and size typical of poxviruses. Helmut Ruska recognised that the previously existing virus systematics based on clinical syndromes and organic manifestations did not reflect a natural, scientifically founded classification. In 1943, based on a solid body of knowledge, he proposed that all viruses - irrespective of their vertebrate, bacterial, or plant hosts - should be systematically classified by morphological criteria, that is, according to intrinsic viral properties.[18] This principle is still valid today in virus taxonomy.[19] On the basis of measurements and counts by electron microscopy, he concluded that viruses were propagated without particle growth and division, unlike bacteria.[18]
Because Helmut Ruska published mostly in German journals, and owing to the isolation of Germany during the Nazi period and World War II, the work of Helmut Ruska did not become widely known. Thus, the American biophysicist Thomas F Anderson (1911-91), who unknowingly repeated parts of the work previously done in Berlin, has written: “In 1940, when I first heard of the electron microscope which was said to have been developed in Germany, it almost seemed to be a hoax perpetrated on the rest of the world by the Nazis. It should be recalled that in 1940 our relations with Germany were so strained that it was difficult to obtain current literature from that country.”[16]
The first electron “magnifying glass” of Ernst Ruska and Max Knoll (1897-1969), constructed in 1929, was a single-magnetic-lens instrument, basically a cathode-ray oscillograph, consisting of a gas discharge tube with cold cathode, an anode, and the specific coil to focus the electron beam and form the image of an object, an annular aperture, on a fluorescent screen. As a prototype, it merely showed the feasibility of the new imaging principle. The next instrument, operational in 1931 (figure 3), was a true microscope, equipped with two electromagnetic lenses, allowing two-stage imaging at a 16-fold magnification.[20] In Berlin at least three research groups were striving for technical progress in the construction of electron microscopes.[21-23] In the mid 1930s, the situation entered a critical phase, since many influential biologists and physicists doubted whether electrons could be used in biomedicine for high-resolution imaging.20 The ability to visualise sub-light microscopic structures - if indeed they existed in living nature - was seriously questioned because a state of continuous change was envisaged, which would in principle prevent these minute structures from being visualised. Even assuming the existence of well-defined supramolecular structures, it was thought that the electron microscope and the necessary preparation techniques would be incapable of furnishing a correct structural analysis of biological objects: the fine structure would be completely destroyed by dehydration of the biological sample in the vacuum of the instrument and would in any case be burnt to ashes in the microscope’s intense electron beam.
The sketch shows the elements of the forthcoming two-stage imaging system that attained 16-fold magnification only 2 months later. During this critical phase, the enthusiasm of the medical doctor Helmut Ruska motivated Ernst Ruska and Bodo von Borries to apply electron microscopy to the elucidation of life and disease processes. With perseverance and mutual encouragement, they managed to overcome many obstacles. Decisive support and arguments in favour of constructing and improving the electron microscope to the point at which it became a functional instrument came from Richard Siebeck (1883-1965), the influential director of the I Medizinische Universitätsklinik of the Charité Medical School. He exhibited both understanding and far sightedness in a professional assessment on Oct 2, 1936, in which he strongly supported the ideas of Helmut Ruska and claimed that electron microscopy might be able to contribute fundamentally to knowledge in normal and pathological anatomy, cell biology, and infection, with particular emphasis on sublight microscopic infectious agents, such as those of smallpox, chickenpox, measles, mumps, and influenza.[20]
Two companies, Siemens and Halske in Berlin and Carl Zeiss in Jena, felt able to take over the economic and technical risks of developing the electron microscope. In 1937, the Siemens company established the Laboratorium für Übermikroskopie in Berlin-Spandau, which supported the close cooperation of Helmut Ruska with his brother Ernst and Bodo von Borries. Here, in 1937-38, two advanced preserial instruments were constructed (figure 4), one of which was to be used exclusively by Helmut Ruska. In 1940, next to this facility, a guest laboratory with four instruments, devoted to biomedical applications and supervised by Helmut Ruska, was also founded.
Irmela Ruska, the widow of Ernst Ruska, remembers that the investigations were usually done at night - for two reasons. First, there was no way to prevent the transmission of mechanical vibrations, which occurred in the factory during working time. Moreover, during daytime, Helmut Ruska was obliged to fulfil his commitments at the clinic, whilst Ernst Ruska and his team concentrated on technical duties. With the first successful applications, however, Helmut was allowed to reduce his clinical obligations. Irmela Ruska vividly remembers making the long journey to the Siemens factory in northwestern Berlin, together with Ernst’s sister Hede von Borries, to bring the men food.
In those early days of electron microscopy, the reliable routine preparation techniques of today, thin sectioning of cells or organs and negative staining or shadowing of particle suspensions, did not exist. In 1934, in his early electron microscopic studies in Brussels (still confined to light microscopic resolution), Ladislaus Marton (1901-79) has shown that treatment with osmium tetroxide can add contrast to biological objects.[24] Unfortunately, owing to lack of funding and the technical limitations of his instrument, Marton was unable to pursue his promising studies. Helmut Ruska’s work concentrated on the characterisation of particle suspensions; for those he developed proper supporting films and described sample application and evaluation in detail.[25] To generate contrast with the small objects, he occasionally applied osmium fumication; shadowing techniques and negative staining were introduced much later by others.
The decision of the Siemens and Halske company to develop the Ruska instrument to higher performance and serial production was doubtless reinforced by the fact that Reinhold Rüdenberg (1883-1961) had patented the principle of electron-microscope imaging in 1931 for Siemens and Halske (even though there was no experimental investigation in the company in 1931). Rüdenberg, as pointed out by his co-worker Max Steenbeck (1904-81), was kept well informed about the experimental achievements of Ernst Ruska and Max Knoll.[26,27] Rüdenberg initially served in the Patent Department and in 1923 became “Chefelektriker” of the company. A case of viral disease in his family might have prompted his interest in technical solutions for the visualisation of small objects.[28] In 1939 Siemens and Halske began serial productions of the transmission electron microscope;[20] and despite the many obstructive regulations and difficulties in war-time Germany, more than 40 microscopes had been built by 1945.[23]
Further use in virus research
One important reason for this rapid development is the many questions that had accumulated in biology and medicine at this time. There was definitely a need for this new instrument, and the developments in electron microscopy and virus research were mutually beneficial. It now became possible to visualise directly what had previously been characterised by indirect techniques, such as filtration and ultracentrifugation for size determination of small particles. The visualisation of virus particles brought reality to the theoretical concepts in virology and thus, led to a thorough understanding of an entirely new world of pathogens.
Later progress in virus research and molecular biology was often based on the use of the electron microscope. Two important discoveries, relating to viruses that ultimately depended on the electron microscope, led to Nobel awards. Wendell Meredith Stanley (1904-71) showed the ability of tobacco-mosaic virus to become crystallised and that the crystals were formed by viral protein. Aaron Klug (1926) used the principles of electron diffraction to unravel the three-dimensional structure of viruses. Today, the field of electron microscopy together with appropriate preparation techniques has attained high technical and scientific standards. It forms a useful tool in modern cell and molecular biology and remains indispensable in the study of virus-cell interactions and in rapid virus diagnosis.[29]
Scientific tradition of the Charité
Helmut Ruska did not restrict himself to research into viruses. Enthusiastic about the new possibilities he studied different phenomena of life during the 1940s. He was not only involved in the elucidation of the structure of glycogen and the process of blood clotting, but also the fine structure of insect muscle, the iridescent skin of earthworms, and plant chlorophyll. In 1948, Helmut Ruska became a professor at Berlin University (renamed Humboldt University in 1949). At the same time he served as head of the department of micromorphology of the German Academy of Sciences in Berlin-Buch and, later on, at the Max-Planck-Society Institute in Berlin-Dahlem. From 1952-58, he headed the department for micromorphology at the New York State Department of Health in Albany and then accepted an invitation from the University of Düsseldorf, Germany, to become director of the Institut für Biophysik und Elektronenmikroskopie.
Helmut Ruska died on Aug 30, 1973, in Düsseldorf. Sadly, he thus missed the possibility of sharing the Nobel prize with his brother Ernst Ruska when the work of the latter received belated acknowledgment in 1986. Helmut Ruska joins other outstanding personalities at the Charité Medical School (founded in 1710 by the Prussian King Friedrich I as both a military and public hospital) and the Berlin University: the pathologist Rudolf Virchow (1821-1902), the bacteriologist Robert Koch, the bacteriologist and virologist Friedrich Loeffler, the immunologist and founder of chemotherapy Paul Ehrlich (1854-1915), and the neurologist Hans-Gerhard Creutzfeldt (1885-1964). Helmut Ruska’s achievements fully justify his presence among this most distinguished group and, at the same time, he can be seen as one of the founders of virus research at the Charité. We can easily imagine how excited he would have been to see where his pioneering efforts 60 years ago have led today, to the remarkable high-resolution information on viruses and their components that are now available thanks to cryoelectron and immunoelectron microscopy.
We thank Peter W Hawkes (Toulouse) and Kenneth Murray (Edinburgh) for critical reading of the paper; and Carla Ruska, Irmela Ruska, Erdman A Ruska, Thomas Ruska, Lotte Lambert, Cilly Weichan, Siemens, and the anonymous referees for helpful advice and documents.
References
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[2] Leeuwenhoek A. Arcana naturae detecta. Delft: H Krooneveldt, 1695/1697.
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[5] Koch R, Schwalbe J, eds. Gesammelte Werke von Robert Koch. Leipzig: Thieme, 1912.
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[7] Beijerinck MW. Over een contagium vivum fluidum als oorzaak van de vlekziekte der tabaksbladen. Versl Gew Verg Wiss en Natuurk Afd, Kon Aka Wetensch Amst 1898; 7: 229-35.
[8] Twort FW. An investigation on the nature of ultra-microscopic viruses. Lancet 1915; ii: 1241-43.
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[11] Abbe E. Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung. M Schultze’s Arch Microsc Anat 1873; 9: 413-68.
[12] von Borries, Ruska E, Ruska H. Bakterien und Virus in übermikroskopischer Aufnahme. Klin Wochenschr 1938; 17: 921-25.
[13] Kausche GA, Pfankuch E, Ruska H. Die Sichtbarmachung von pflanzlichem Virus im Übermikroskop. Naturwissenschaften 1939; 27: 292-99.
[14] Ruska H. Über das Virus der Varicellen und des Zoster. Klin Wochenschr 1942; 22: 703-04.
[15] Ruska H. Die Sichtbarmachung der bakteriophagen Lyse im Übermikroskop. Naturwissenschaften 1940; 28: 45-46.
[16] Cairns J, Stent GS, Watson JD, eds. Phage and the origins of molecular biology. New York: Cold Spring Harbor Laboratory Press, 1966.
[17] Zaitlin M. Tobacco mosaic virus and its contributions to virology. ASM News 1999; 65: 675-80.
[18] Ruska H. Versuch zu einer Ordnung der Virusarten. Arch ges Virusforsch 1943; 2: 480-98.
[19] Murphy FA, Fauquet CM, Bishop DHL, et al. Sixth Report of the International Committee on Taxonomy of Viruses (ICTV): classification and nomenclature of viruses. Arch Virol Suppl 10. Wien, New York: Springer, 1995.
[20] Ruska E (translated by Mulvey T). The early development of electron lenses and electron microscopy. Stuttgart: Hirzel, 1980.
[21] Brüche E, Haagen E. Ein neues, einfaches Übermikroskop und seine Anwendung in der Bakteriologie. Naturwissenschaften 1940; 28: 113-27.
[22] von Ardenne M. Ergebnisse einer neuen Elektronenmikroskopanlage. Naturwissenschaften 1940; 28: 113-27.
[23] Wolpers C. Electron microscopy in Berlin 1928-1945. Adv Electronics Electron Phys 1991; 81: 211-29 [PubMed].
[24] Marton L. Electron microscopy of biological objects. Nature 1934; 133: 911.
[25] Ruska H. Übermikroskopische Untersuchungstechnik. Naturwissenschaften 1939; 27: 287-92.
[26] Hawkes PW. Complementary accounts of the history of electron microscopy. Adv Electronics Electron Phys 1985; suppl 16: 589-618.
[27] Ruska E. The emergence of the electron microscope: connection between realization and first patent application, documents of an invention. J Ultrastruct Molec Struct Res 1986; 95: 3-28 [PubMed].
[28] Rüdenberg R. The early history of the electron microscope. J Appl Phys 1943; 14: 434-36.
[29] Biel SS, Gelderblom HR. Diagnostic electron microscopy is still a timely and rewarding method. J Clin Virol 1999; 13: 105-19 [PubMed].
This text ist published by permission of the authors and The Lancet.