Rudolf Rigler

Torbjörn Caspersson

JUBILEUMS E S S Ä E R

- the founder of cellular biophysics

Rudolf Rigler Essäist

Torbjörn Caspersson - the founder of cellular biophysics Introduction Torbjörn Caspersson was born 1910 in Motala an industrial town situated at lake Vättern in Middle Sweden. He decided to join the Karolinksa Institutet in Stockholm for studying medicine. Here he met Einar Hammarsten a portal figure in the chemistry and biochemistry of nucleic acids, a field which had been pioneered by FriedrIch Miescher in Basel and Albert Kossel in Heidelberg..

Torbjörn Caspersson and Einar Hammarsten, were following up the polymeric nature of nucleic acids which was believed at that time to consist of tetranucleotides. In collaboration with Rudolf Signer in Bern Caspersson and Hammarsten gave evidence for the polymeric structure of nucleic acids. (Signer.

Caspersson & Hammarsten, 1938).

The fundamental way of Hammarsten in discovering the nature of biochemical processes thoroughly influenced Torbjörn Caspersson and he was fascinated by the idea to analyze nucleic acids within the cellular environment. For this he envisaged spectroscopic methods in particular the UV absorption of nucleic acids and proteins as the right approach.

Ultraviolett spectroscopy of Cellular Components. Jena and August Köhler In Jena at Carl Zeiss in the beginning of 1900 August Kohler pursued the legacy of Ernst v.Abbe by improving the resolution of the microscope introducing ultraviolet transmitting optics as well as the Köhler illumination principle Together with Moritz von Rohr he developed the first ultraviolet microscope. (Köhler & von Rohr, 1904).This was the scenario which drew Torbjörn Caspersson to come to Jena and to collaborate with August Köhler.

In Stockholm at the basement of the chemistry department of the Karolinska Institutet at Hantverkargatan in Kungsholmen (known as potatiskällare to Torbjörns collaborators) an ultraviolet microscope was built up which could measure the UV absorption of nucleic acids and proteins at specific wavelengths in individual cells.

For generation of UV light which had to be focused into the system a large spark gap generator was used. Since appropriate detectors at this time were not accessible photographic plates were used to measure the UV absorption at wavelengths specific for nucleic acids (260 nm) and proteins (280 nm).

At the wavelengths for which the microscope objectives had been optimized the photographic plates were exposed to the transmitted UV radiation The information stored in the photographic plates was then transformed into quantitative spectral data by a plate reader which Caspersson received from Manne Siegbahn who had received the Nobel prize in Physics (1924).

When I joined Torbjörn in 1960 Lennart Aquilonius (“Quille”), Caspersson´s first engineer introduced me to the original setup from Hantverkargatan which was placed in a dark room like before.

This instrument formed the basis of Torbjörn Caspersson´s first important and pioneering work (Caspersson, 1936).

His thesis published in Acta Physiologica Scandinavica 1936 was a landmark in quantitative biological chemistry and cellular biology. He soon was joined by Jack Schultz who had graduated with Thomas Hunt Morgan from Caltech. Together they started the ground breaking work on the relationship between nucleic acid turnover and production of proteins in the Balbiani rings of Drosophila chromosomes (T. Caspersson & J. Schultz, 1938. 1939).

I myself had the opportunity to visit Jack

Schultz in 1967, at that time director of the Fox Chase Cancer Institute in Philadelphia and I still remember Jacks recollection of his time with Torbjörn in the late thirties. The development of quantitative ultraviolet microspectroscopy had an immediate impact on several fields of importance to medicine like nucleic acids in tumor growth (Caspersson & Santesson, 1942), in bacterial growth (Malmgren & Hedén, 1947) and in formation of blood cells (Theorell, 1947).

Collaborators of this time were Holger Hydén, who later became professor of histology at Gothenburg university and Arne Engström who became the first professor of Medical Physics at the Karolinska Institute. Hyden was the founder of cellular neurochemistry and Engström pioneered cellular X-ray analysis and microradiography.

The fundamental work of Torbjörn Caspersson between 1936 and 1952 was summarized in his monographs on “Cell growth and Cell function” (1952) which showed the correlation between production of RNA and proteins as was evident from the Drosophila experiments. His contemporary Jean Brachet (1909-1886) who like Caspersson had a medical background contributed to Caspersson´s observation suggesting that RNA production apparently was linked to protein synthesis (Brachet, 1942). At this time the genetic work flow from DNA to RNA to protein was yet unclear, however one year later in 1953 Francis Crick and James Watson unveiled the secret in their paper on the structure of DNA. It was the start of Mole-

cular Biology in combining structural information and biochemical analysis of protein synthesis.In fact James Watson was supposed to join Torbjörn Caspersson but finally decided to go to Cambridge where he met Francis Crick.

The Medical Nobel Institutet for Cell Research in Solna Torbjörn Caspersson was promoted to professor at the Karolinska Institutet 1944 and became head of the Medical Nobelinstitutet for Cell Research established at the new campus of the Karolinska Instutet in Solna (1952). Together with the Nobelinstitute for Biochemistry built for Hugo Theorell it was located in the building adjacent to the Nobel Forum. Both Institutes had a common library with the most exquisite collection of scientific literature in biosciences of its time.

When I joined Torbjörn Caspersson in 1960. I realized that the Institute for Cell Research was a physics department with the aim to unravel the cellular functions by biophysical methods at a level which even today would be considered as extraordinary. Underneath the mechanical workshops in the ground floor several generators were placed which could deliver electric currents of any voltage and frequency to every laboratory of the department. The mechanical workshops in which the new UV spectrographs were built had the most exquisite tools for high precision machining.This work which reached the µm precision level was done by Rune Säfström and Sigurd Carlsson The engineering and construction was in the hand of Gösta Lomakka an expert in electronic engineering. He was supported by Leon Carlson who came from optical physics at the KTH. Gösta was one of the most ingenious persons I met and he realized TCs* ideas by inventing and constructing the scan-

ning UV microspectrometers. At a later time as head of the department of Medical Physics I had the previlege to work with Gösta who joined me after Torbjörn Caspersson´s retirement. Now involved in fluorescence correlation spectroscopy (FCS) which I started in Göttingen and continued with Måns Ehrenberg, Gösta helped us to realize the equipment which finally led to the image of single molecules.

Scanning UV microspectrophotometry The step from the analysis of cell components by UV imaging with photographic plates to the quantitative measurement of UV absorbing material in individual parts of a cell required three important developments: (i) introduction of sensitive photo detectors, (ii) solving the problem caused by light scattering from cellular objects which was not related to UV absorption (Mie scattering) (iii) wavelength dependent image focusing of UV objectives (chromatic aberration).

The first requirement was solved by the introduction of photomultiplier detectors which were developed by RCA after the war. TC came back from the USA with the RCA 1P21 which was introduced in the second UV spectrograph called “Tvåan” the second, a huge instrument which filled a whole room. It was a double beam instrument which had the detectors placed several meters away from the UV objective in order to ensure small measurement areas necessary for scanning small objects. The scanning procedure was necessary to overcome the problems of Mie scattering which overshadowed the signal due to UV absorption of cellular material.

Gösta Lomakka invented and constructed for this purpose a hysteresis free scanning system which moved the sample with µm precision along a line.The signal was recorded in an analog recorder on paper. For achieving a 2 dimensional scan

the scanning line was moved mechanically by a mirror. Finally a recording of the UV absorption line by line for a single cell could be obtained, a procedure which generated meters of paper in an hour and had to be evaluated by hand.

This in contrast to the later UV spectrograph Trean, Fyran, Femman och Sexan which allowed automatic scanning and computation of the UV absorption from the whole scanned area within seconds.

One has to remember that digital electronics did not exist at this time and all data had to be recorded by analog electronic devices. This was accomplished by Gösta Lomakka´s extraordinary skills.

A most important development were the wavelength corrected UV optics. The spirit of August Köhler, Torbjörn Caspersson´s mentor, had still its influence. Köhler’s collaborator at Zeiss, Kurt Michels became head of research at the Zeiss Company which moved after the war from Jena to Oberkochen in West Germany. Under Michels guidance the UV Ultrafluar objectives were developed which had the unique property of a wavelength corrected focus which extended over several hundred µm.

This was the background of the UltraMicroSpectrophotometer UMSP developed and produced by Zeiss in collaboration with Torbjörn Caspersson and Gösta Lomakka.It was probably the most exquisite optical instrument of its time and a large number of instruments were produced and used in many laboratories worldwide. At the Cell Research 2 instruments existed of which one still is at the Karolinska Institutet (MBB). The UMSP became later the background of the fluorescence microspectrograph. I still remember collaborators of Harold Urey using the UMSP for analyzing meteorite material for finding signs of (previous) living material by recording UV spectra.

Trean was the working horse for UV spectroscopy of cells cultured in vitro and ge-

nerated the important data on the nucleic acid reduplication under cell growth as demonstrated in the theses of Dick Killander and Anders Zetterberg .Other names were Burt Gladhill analyzing the meiotic cell division during sperm maturation or Gerd Auer in cell division and tumor formation. The majority of TCs students and collaborators became known professors at the Karolinska Institutet.

Interferometric mass determination, the balance of cellular analysis The first way to determine the mass of cellular components was based on soft X-ray absorption an energy range were the Xray absorption coefficient is almost identical for the important atoms involved in biological compounds, C, O, N, H. The first soft X-ray instrument for mass determination was built by Arne Engström and Bo Lindström. It provided the basis for a reference for optical mass determination.

These instruments are based on the interferometrically determined optical path length for a light beam transversing the cell component in question. Also these instruments which got the name Johanna and Johannita instead of numbers had the scanning principle incorporated and constituted highly complex optical machines.

For quantitative analysis the refraction index of proteins and nucleic acids had to be known and were determined with high precision by Leon Carlson. Together with UV absorption and dry mass analysis a very detailed picture of the main constituents and their change during the cell cycles could be obtained.

In the laboratories of Cell Research one of the early electron microscopes from RCA was situated. It was used by Elmar Zeitler and Gunther Bahr for developing a method for mass determination of biological matter by electron absorption (Zeitler and Bahr 1957). I met Elmar later when he was head of the Max Planck Institute of

Electron Microscopy in Berlin and Gunter at the Armed Forces Pathology Institute in Washington.

Fluorescence Microspectrometry My interaction with Torbjรถrn Caspersson in the first visit in 1960 was a learning phase while the second visit which started in 1962 turned out to be a life long interaction with the Karolinska Institutet.

TC knew about my biophysical interests and my education at the Department of Physics in Graz with Adolf Smekal who predicted the Raman effect. He proposed to me to develop a fluorescence microspectrograph which I accepted instantaneously. Putting my medical interests aside I only had an eye for the physics part of this project. The UMSP became the basis for the fluorescence microspectrometer. At this time fluorescence microsopy was well known. However nobody thought that taking fluorescence spectra from tiny and bleakly fluorescent objects would be possible. The final outcome however showed that the emission from small object such as mitochondria could be measured (Rigler,1966). I used for this purpose the fluorescent dye Acridin Orange which had a high affinity to nucleic acids and polyanions (Rigler,1967). The sensitivity of the instrument was high enough to detect the Acridine Orange fluorochrome in individual mitochondria which later turned out to be due to mitochondrial DNA. The ability to detect and measure individual chromosomes laid the ground for the study of the Quinacrin banding of human chromosomes by T. Caspersson and L. Zech (1973) a theme which occupied TCs last time at the Cell Research and provided a strong input into human genetics.

Outlook The development of UV microspectroscopy can be seen as the goal to unravel the secrets of living matter at the cellular

level by biophysical methods.Even if the tools of Molecular Biology in particular Xray crystallography and NMR spectroscopy have been at the center of interest and are contributing to our understanding in a central way, they are unable to solve functional relationships between production, transport and action at the cellular level. What was started by UV absorption spectroscopy has today been taken over by fluorescence spectroscopy of single cells. The introduction of fluorescent proteins and their expression when coupled to distinct genetic products provides highly sensitive and selective markers for understanding detailed processes of the cellular metabolism. Fluorescence Correlation Spectroscopy (FCS) which had been started in my time with Manfred Eigen in Gรถttingen has been strongly influenced by the developments at Cell Research. In particular the microscope based confocal analysis which now has reached the cell level allows to detect individual bimolecular and their pathway.

Carl Zeiss now in Jena again has produced the Confocor 1, 2 and 3 spectrometers and has provided the tools for the studies of cellular dynamics at the level of single bimolecular (Rigler, 2009). A new area of cellular biophysics which has been started by Torbjรถrn Caspersson is ahead of us.

Two events should be remembered. TCs 80th birthday for which Nils Ringertz, then secretary of the Nobel assembly, arranged a common party in the rooms of the old Cell Research with all collaborators from the Cellforskning time and the 85th birthday which was arranged by Gerd Auer at the Cancer Research Building at Karolinska Sjukhuset.

It was a grey December day in 1997 when we had to say farewell to Torbjรถrn at Rรถnninge kyrka. A true pioneer had left us.*

* I have used Torbjörn’s well known signature TC for abbreviation.

References A. Köhler, & M. von Rohr, Z. Instrumentenk. xxiv, 274, (1904).

R. Signer, T. Caspersson L E. Hammarsten, Nature 141, 122 (1938).

T. Caspersson, Über den chemischen Aufbau der Strukturen des. Zellkernes.” Acta Physiolog.Scand. (1936), T. Caspersson & J. Schultz, Nucleic Acid Metabolism of the Chromosomes in Relation to Gene Reproduction Nature 142, 294, (1938).

T. Caspersson & J. Schultz. Pentose Nucleotides in the cytoplasm of growing tissues ,Nature 143, 602, 1939.

J. Brachet, La localization des acids pentosenucléiques dans les tissus animeaux et d´ les oeufs amphibiens en voie de developpments Archives de biologie 53, 207, 1942.

T. Caspersson & L. Santesson, Studies on protein metabolism in the cells of epithelial tumours. Acta radiol., Stockh. 46: 113, (1942).

B. Malmgren & C. G. Heden Nucleotide Metabolism of Bacteria and the Bacterial Nucleus. Nature 159, 577, (1947).

B. Thorell, Studies on the formation of cellular substances during blood cell production. Thesis Karolinska Institutet, Stockholm (1947).

T. Caspersson, Cell Growth and Cell Function W, W, Norton New York ( 1952).

E. Zeitler & G. F. Bahr Exp. Cell Res.12, 44 (1967).

R. Rigler, Microspectrofluorometric characterization of intracellular nucleic acids and nucleoproteins by Acridine Orange, Acta Physiol. Scandn (1966).

R. Rigler, Acridine Orange in Nucleic Acid Analysis, Annals of the New York Academy of Sciences, vol. 157, 211, (1967).

T. Caspersson & L.Zech, Nobel Symposium No, 73 Chromosome Identification Technique and Application in Biology and Medicine (1972).

R. Rigler, FCS and Single Molecule Spectroscopy. In Nobel Symposium 138, Single Molecule Spectroscopy in Chemistry, Physics and Biology, ed. A. Gräslund, R. Rigler & J. Widengren Springer 2009.

Rudolf Rigler, Essäist TORBJÖRN CASPERSSON född 1910 i Motala. Medicinstudier vid Karolinska Institutet.Elev till Einar Hammarsten. Första arbeten över DNA molekylers lösningsstruktur. Samarbete med A. Köhler i Jena, UV optisk bildanalys för spektroskopisk bestämning av cellulära nukleinsyror och proteiner.

RUDOLF RIGLER, född 1936 i Frankfurt/ Main Medicinstudier i Graz och Frankfurt/ Main, Dr,med, Universitet Graz 1960.

Disputation 1936, Professor vid Karolinska Institutet 1944.

Samarbete med Manfred Eigen (Nobelpris kemi 1967) 1967.

Nobelinstitutionen för Med. Cellforskning och Genetik 1952.

Rådsprofessur i molekylär biofysik vid KI 1976, Professor i molekylår biofysik vid Lunds universitet 1983-1985, Professor i Medicinsk Fysik vid KI 1985-2001.

Jahrespris 1971, Balzanpris 1979 UV-MIcrospectrophotometry, Chromosome banding.

Samarbete med Torbjörn Caspersson 1960-1997.

Disputation Karolinska institutet 1966.

Professeur invité Swiss Federal Institute of Technology Lausanne (EPFL) 2001 Development of Fluorescence Microspectrophotometry.

Development of Fluorescence Correlation Spectroscopy (FCS).