Research Groups - Max-Planck-Institut für biophysikalische Chemie

Research Groups
The following pages feature the wide spectrum represented
by the institute’s research program and groups.
In-depth Looks
Without X-ray structural analysis, Francis Crick and James Watson would not have
discovered that DNA, our prime genetic information carrier, has a double helical
structure. And how would have Robert Koch – also a Nobel Laureate – discovered the
virus that causes anthrax without having a good microscope at his disposal? High-end
science requires high-end equipment. Therefore, it is no wonder that so many scientists
are involved in methodical innovations. For example, it takes new spectroscopic and
microscopic procedures to determine structural details at the single molecule level as
well as to explore the dynamics of molecular processes.

NanoBiophotonics
Dr. Stefan W. Hell
Stefan Hell received his doctorate
in physics from the University of
Heidelberg in 1990. From 1991 to
1993, he worked at the European
Molecular Biology Laboratory in
Heidelberg, followed by a 1993 to
1996 stay as a senior researcher at the
University of Turku (Finland), and as a
visiting scientist at Oxford University
(England). In 1996, he received his
›Habilitation‹ from the University of
Heidelberg, where he teaches physics.
In 1997, he joined the Max Planck
Institute for Biophysical Chemistry as a
junior Max Planck research group
leader. In 2002, he was appointed
director at the Max Planck Institute for
Biophysical Chemistry, where he heads
the newly established Department of
NanoBiophotonics.
Stefan Hell is the recipient of the Prize
of the International Commission for
Optics (2000), a co-recipient of the
Helmholtz Prize (2001), and of
the Leibinger Innovation award.
In 2002, he received the
Carl Zeiss Prize and the
Karl Heinz Beckurts Prize.
shell@gwdg.de
Bewersdorf, J., R. Pick and S. W. Hell: Multifocal
multiphoton microscopy. Optics Letters 23 (9),
655–657 (1998).
Hell, S.W. and M. Nagorni: 4Pi-confocal
microscopy with alternate interference. Optics
Letters 23, 1567–1569 (1998).
Klar, T.A., S. Jakobs, M. Dyba, A. Egner and
S.W. Hell: Fluorescence microscopy with diffraction
resolution barrier broken by stimulated
emission. Proc. Natl. Acad. Sci. USA 97,
8206–8210 (2000).
Egner, A., S. Jakobs and S.W. Hell: Fast 100-nm
resolution three-dimensional microscope
reveals structural plasticity of mitochondria in
live yeast. Proc. Natl. Acad. Sci. USA 99,
3370–3375 (2002).
Dyba, M. and S.W. Hell: Focal spots of size
λ/23 open up far-field fluorescence microscopy
at 33 nm axial resolution. Phys. Rev. Lett. 88,
163901–163901 (2002).
Would it not be great to observe the cell organelles and
the myriad of proteins in a living cell in 3D with
nanoscale resolution and with a non-invasive
method? Until recently, devising or even building such a microscope
had been a fundamental scientific problem. The reason for
this is that a non-invasive, high-resolution microscope needs to
rely on focused visible light. Fluorescently labeled proteins, nucleic
acids, and lipids can be mapped in a living cell with unmatched
sensitivity. However, the established light microscopy methods suffer
from a critical disadvantage, namely their diffraction-imposed
resolution, which is limited at best to ~500 nm in the direction of
light propagation (z-axis) and ~200 nm in the transverse (xy)
direction. The diffraction barrier was discovered by Abbe in 1873
and has been paradigmatic ever since.
We have recently succeeded in introducing physical concepts that
break Abbe’s barrier. In fact, we intend to explore all the possibilities
of making the microscope sharper. Our primary scientific aim
is to push fluorescence microscopy resolution down to the
nanometer range and to eventually apply it to the field of biology.
This challenging endeavour requires a joint effort of physicists,
chemists, engineers, and biologists alike.
Perhaps the most intuitive method for sharpening the spot is to
selectively inhibit the fluorescence at the circumference by stimulated
emission, a natural competitor to fluorescence. Technically,
the Stimulated Emission Depletion (STED-) microscope relies on
pairs of synchronized laser pulses. The primary excitation pulse
produces a 3D spot of excited molecules of regular diffraction size,
and the immediately following red-shifted STED pulse quenches
the excited molecules down to the ground state. By arranging the
STED pulse in a doughnut mode, only the fluorescence at the
periphery of the spot is inhibited, whereas the very centre ideally
remains untouched. To date, an improvement of factor 3 in the
transverse direction and up to 6 along the optic axis has been
demonstrated (Fig.1). In a similar manner, it is possible to deplete
the ground state of the fluorophore. Both concepts rely on the nonlinear
optical phenomenon of saturation and have the potential to
provide resolution down to the molecular scale.
A powerful way of sharpening the axial resolution of a microscope
is the coherent superposition of focal wavefronts. This idea has
been realized in our 4Pi-microscope for which we use two opposing
objective lenses, adding up their wavefronts to form a 3- to 7-fold
improvement in spatial resolution along the optic axis. The resolution
is further enhanced by the application of (non-linear) image
18 departments and research groups
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estoration techniques (Fig.2). The
superior 3D-resolution of a 4Piconfocal
microscope is illustrated in
Fig.3, which depicts a 3D-rendered
image of the mitochondrial network
of a live budding yeast cell. The
combination of both microscopes,
namely the STED-4Pi-microscope,
was the first to demonstrate a spatial
resolution in the double-digit nanometer
range (30-40 nm) with visible
light and regular objective lenses.
Fig. 1. Stimulated emission depletion microscope. The diffraction-limited
distribution of excited molecules generated by the green beam is depleted
at the periphery through stimulated emission with the red beam (a). The
resulting fluorescence spot (b) is fundamentally reduced as compared to its
standard counterpart (c).
Fig. 2. Optical section-image through the microtubules of a fixed mousefibroblast
cell, labeled by immunofluorescence. In conjunction with image
restoration, 4Pi-confocal microscopy provides a 3D-resolution in the range
of ∼100 nm.
Fig. 3. 3D-rendered image of the mitochondrial
network of a live yeast cell. The mitochondrial
matrix is labeled with green fluorescent protein,
whereas the cell wall is counterstained with the dye
calcofluor white and displayed by ray tracing.
Live cell 4Pi-imaging with ∼100 nm 3D-resolution
allowed us to study the influence of selected
mitochondrial proteins on mitochondrial
morphology.
departments and research groups 19

Spectroscopy and
Photochemical Kinetics
Professor Hans-Jürgen Troe
Hans-Jürgen Troe received his
doctorate at the University of
Göttingen in 1965, where he also
completed his ›Habilitation‹ in
physical chemistry in 1967. In 1971,
he was appointed Full Professor for
Physical Chemistry at the École
Polytechnique Fédérale de Lausanne.
In 1975, he returned to Göttingen as
director of the Institut für Physikalische
Chemie of the University. Since 1990, he
has also been director of the Department
of Spectroscopy and Photochemical
Kinetics at the Max Planck Institute for
Biophysical Chemistry.
Hans-Jürgen Troe has received honorary
doctorates from the Universities of
Bordeaux and Karlsruhe, and he is an
Honorary Professor of the École
Polytechnique Fédérale de Lausanne. In
addition, he has received many awards, is
a member of several scientific academies
and currently serves as a member of
the Senate of the Deutsche
Forschungsgemeinschaft.
Internal research groups:
Prof. Walter Hack,
Dr. Günter Käb
Dr. Thomas Lenzer
Prof. Klaus Luther
Dr. Derek Marsh
Prof. Jörg Schroeder
Prof. Dirk Schwarzer
Dr. Simone Techert
Dr. Max Teubner
Dr. Jomo Walla
Dr. Klaas Zachariasse
Independent research group:
Prof. Michael Stuke
Contact person:
Dirk Schwarzer
dschwar@gwdg.de
Many phenomena in the animate and inanimate realms
of nature can be traced back to molecular processes. In
the atmosphere, molecules are continuously created by
collisions of reactive particles, while absorption of light or heat
energy makes them dissociate once again. Surprisingly, these
atmospheric reactions resemble those occurring both in flames and
in internal combustion engines. Solvents play an important role in
chemical reactions in fluid environments, whereas molecular packing
density is the decisive factor for solid-state reactions. For larger
molecules, which include such biopolymers as proteins and
nucleic acids, internal molecular dynamics make an additional
contribution to the interactions between single molecules.
The time dependence of chemical reactions, also known as reaction
kinetics, is determined by intermolecular interactions as well
as by processes occurring within the molecule itself. This ensures
that the energy transfer is used to effect changes in the structure,
and thus, the function of the molecule. Therefore, the visualization
of molecular processes is an important prerequisite for understanding
nature.
Molecular spectroscopy enables us to take this close-up look at
nature: thus, it is possible for us to identify different molecular
species, but also those that differ merely in structure or energy
An iodine molecule is split with a
laser pulse; 100 picoseconds later,
the diffraction pattern of an x-ray
pulse reveals how the iodine
atoms manage to recombine.
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state, by their spectra. In addition, molecules can be
excited photochemically by the absorption of light
energy. This procedure makes it possible for us to
follow the photochemical kinetics by spectroscopy:
one pulse of light initiates the reaction, and a second
pulse interrogates the response.
A main focus is the dynamics of chemical reactions in
different environments: We study this phenomenon in
isolated molecules as well as in molecular aggregates,
gases, supercritical fluids and liquids, and also in
crystals and membranes. The experiments are conducted
under a wide range of conditions – temperatures
vary from a few degrees absolute up to
thousands of degrees, pressures range from a few
thousandths of an atmosphere up to several thousand
atmospheres, and we also work with high magnetic
fields and different types of solvents. This enables us
to unravel the various interdependent factors that
play a part in the course of a chemical reaction. Not
only do the spectra make it possible to distinguish different
molecules and their energy states, they also
yield important information about the effect of the
surroundings. Modern experimental methods permit
us to achieve a time resolution that extends down to
about ten femtoseconds (the one hundred-millionth
part of a millionth of a second). This is the timescale
on which the motions of atoms in molecules occur. In
this way, the internal dynamics of a molecule can be
›seen‹ directly. Numerical simulations allow the observed
phenomenon to be reproduced by molecular
dynamics on the computer.
In molecular rearrangements, the crossing of energy
barriers is of central importance: in the transition
from reactants to products of a chemical reaction,
often such a barrier must be overcome first, rather
like crossing a mountain pass between two valleys.
The height of the barrier may be modified either by
specific structural changes in the molecule or by its
Diiodomethane in chloroform
– snapshot from a molecular
dynamics computer simulation
(iodine atoms: violet; chlorine
atoms: orange).
departments and research groups 21

environment. In this process, the solvent can act as a
viscous medium and oppose the crossing of the
barrier. The dynamics of such events can be followed
directly with ultra short light pulses.
Our group also investigates the transport of electrons,
protons and hydrogen atoms within the molecule.
Time-resolved absorption and emission spectroscopy
allow us to study the influence of
structure and
energetics on
fluorescence
quenching,
on charge
separation,
of spin-labeled biomolecules it is possible to study
dynamic processes in membranes, lipid-protein interactions,
and essential aspects of membrane assembly
in considerable molecular
detail.
Molecular structures
can also
be determined
by high-resolution
x-ray
diffraction
methods.
Such experiments
are
made with a
pulsed x-ray
beam that is
Laser spectroscopy
allows the study of
energy transport:
The dye azulene
(red) is excited by a
laser pulse. Within
four picoseconds a
large part of the
absorbed energy is
transmitted to the
anthracene chromophore
(blue) via
the hydrocarbon
chain (green).
and charge recombination. To know the mechanism
for fluorescence quenching in detail is one of the
essential prerequisites for the use of fluorescent dyes
as probes in biological systems, such as in the analysis
of DNA.
Biochemical reactions in cell membranes are also controlled
by molecular dynamics. Electron spin resonance
spectroscopy is a highly sensitive biophysical
tool for the study of these processes. Through the use
synchronized with the light pulse that starts the
photochemical reaction. Pilot studies with simple
reactions in the liquid or solid state have yielded
promising results.
Innovative experiments, numerical simulations and
theoretical studies of the physicochemical principles
of molecular processes complement each other in our
aim of reaching a quantitative understanding of
molecular dynamics.
Marsh, D., and L.I. Horváth: Structure, dynamics and composition of the
lipid-protein interface. Perspectives from spin-labelling; Biochim. Biophys.
Acta 1376, 267 (1998).
Heidelbach, C., V.S. Vikhrenko, D. Schwarzer, I.I. Fedchenia and J. Schroeder:
Molecular dynamics simulation of vibrational energy relaxation of highly
excited molecules in fluids. III. Equilibrium simulations of vibrational energy
relaxation of azulene in carbon dioxide. J. Chem. Phys. 111, 8022 (1999).
Harding, L.B., A.I. Maergoiz, J. Troe, and V.G. Ushakov: Statistical rate theory
for the HO + O ⇔ HO 2 ⇔ H + O 2 reaction system: SACM/CT calculations
between 0 and 5000 K. J. Chem. Phys. 113, 11019 (2000).
Charvat, A., J. Aßmann, B. Abel, D. Schwarzer, K. Henning, K. Luther and J.
Troe : Direkt observation of intramolecular vibrational energy redistribution of
selectively excited CH 2 I 2 and C 3 H 5 I molecules in solution. Phys. Chem. Chem.
Phys. 3, 2230 (2001).
Daum, R., S. Druzhinin, D. Ernst, L. Rupp, J. Schroeder, and K.A.
Zachariasse : Fluorescence excitation spectra of jet-cooled 4-(diisopropylamino)benzonitril
and related compounds. Chem. Phys. Lett. 341, 272
(2001).
Neutze, R., R.Wouts, S.Techert, A.Kirrander, J.Davidson, M.Kocsis, F.Schotte,
and M.Wulff : Visualizing photochemical dynamics in solution through
picosecond x-ray scattering. Phys. Rev. Lett. 87, 195508 (2001).
22 departments and research groups www.mpibpc.mpg.de/abteilungen/010/

Biochemical Kinetics
Alack of perfection during the reproduction process – that
is the main reason for the fascinating variety of life
forms on earth. Although DNA, our genetic information
carrier, is an extremely stable macromolecule, errors sometimes do
occur when information is copied and duplicated. Since not all of
these errors are corrected and repaired, new variants emerge all the
time. These variants form the basis for the evolution of organisms.
All living species are subject to these transformations.
We can define evolution as a natural phenomenon, but we may
also re-enact it in a controlled setting to meet our own needs. In
these experiments, bacteria, viruses, or single nucleic acid molecules
are encouraged to reproduce, and then selected according to
specific criteria. For this purpose, we have developed special
equipment that can process many thousand samples simultaneously.
In this way, we can study basic mechanisms of evolution,
e. g. the tricks that HIV and other cunning pathogens use to outsmart
our immune system. Moreover, such »evolution machines«
can help developing the molecular agents needed for novel drugs.
These different applications have one crucial aspect in common: In
all of these cases, tiny quantities of substances need to be analyzed.
This amounts to »finding a needle in the haystack.« It also
goes for diagnostic applications, such as the early diagnosis of BSE.
Special spectroscopic methods enable us to detect even single molecules.
This has lead to the development of evolutive biotechniques
that permit the recognition of single biomolecules in a specific
distribution as well as the optimization of their function.
Professor Manfred Eigen
Manfred Eigen received his doctorate
from the University of Göttingen in 1951,
where he subsequently worked at the
Institute of Physical Chemistry. After joining
the Max Planck Institute for Physical
Chemistry in 1953, he became director
of its Chemical Kinetics department in
1964. On Professor Eigen’s suggestion,
this institute became part of today’s
Max Planck Institute for Biophysical
Chemistry in 1971, where he has been
director of the department of
Biochemical Kinetics. Even after his
retirement in 1995, he has been
active to the present day. In 1967,
Manfred Eigen was awarded
the Nobel Prize for Chemistry
for his research on ultra-fast
chemical reactions
in solutions.
Eigen, M.: Molekulare Diagnostik. In: Das Gen
und der Mensch. (Ed.) G. Gottschalk.
Wallstein-Verlag, Göttingen, 2000, 137–167.
Eigen, M.: Natural selection: a phase transition?
Biophys. Chem. 85 (2-3), 101–123 (2000).
Koltermann, A., U. Kettling, J. Bieschke, T.
Winkler, M. Eigen: Rapid assay processing by
integration of dual-color fluorescence crosscorrelation
spectroscopy: High throughput screening
for enzyme activity. Proc. Natl. Acad. Sci.
USA 95, 1421–1426 (1998).
Eigen, M.: Steps towards life, a perspective on
evolution. Oxford University Press, Oxford,
1992.
Brakmann, S., U. Kettling, und F. Oehlenschläger:
Die Evolutive Biotechnologie und ihre
Perspektiven. Biologie in unserer Zeit 6,
355–366 (1995).
Automated evolution and screening device of DIREVO Biosystems AG/
Cologne, a biotech company that grew out of the Department of
Biochemical Kinetics at the Max Planck Institute for Biophysical Chemistry.
www.mpibpc.mpg.de/abteilungen/081/
departments and research groups 23