FSM 2015 - International Biophysics Conference

FSM 2015 - International Biophysics Conference
Fluorescence Spectroscopy, Microscopy and Molecular Cell
Mechanics
Program and Abstract Book
8th – 11th October 2015
Pollentia Club Resort, 07400 Alcúdia, Spain
Event organizer:
Georg August University
Third Institute of Physics
Friedrich Hund Platz 1
37077 Göttingen, Germany

Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics
2

3
Meet our sponsors and partners
The FSM 2015 organization committee is grateful for the support
of our sponsors and partners:

4
Scientific program
Thursday 08.10.2015
17:00 - 17:10 Welcome message
Session: Single molecule spectroscopy biophysics I Chair: Prof. Dr. Sauer
17:10 - 17:40 Prof. Dr. Robert Ros, ASU Tempe, Arizona, USA (Invited Talk)
Physical Properties of Fibrinogen Matrices Control the Adhesion of Blood Cells
17:40 - 18:00 Marina Dietz, Frankfurt, Germany (Student Award)
Single-molecule studies provide new insights into MET receptor activation
18:00 - 18:20 COFFEE BREAK
Session: Single molecule spectroscopy biophysics II Chair: Prof. Dr. Schmidt
18:20 - 18:50 Prof. Dr. Jörg Langowski, Heidelberg, Germany (Invited Talk)
Chromatin dynamics are controlled by nuclear lamin A
18:50 - 19:10 Kathrin Tegeler, Heidelberg, Germany (Student Award)
Single molecule fluorescence studies of nucleosome dynamics
19:10 - 19:30 Dr. Katalin Tóth, Heidelberg, Germany
Colorful nucleosomes
19:00 – 20:00 POSTER SESSION
20:00 - … DINNER

5
Friday 09.10.2015
Session: Biophysics of cells Chair: Prof. Dr. Robert Ros
9:00 - 9:30 Prof. Dr. Christoph Schmidt, Göttingen, Germany (Invited Talk)
Active Matter in Cells
9:30 - 9:50 Dr. Florian Rehfeldt, Göttingen, Germany
Cell-Matrix-Mechanics Dictates Stem Cell Fate via Cytoskeleton Structure
9:50 - 10:10 Dr. Renata Garces, Göttingen, Germany
Mechanosensitive channels in bacteria gated by a mechanical load
10:10 - 10:30 Florian Schlosser, Göttingen, Germany
Force fluctuations in three-dimensional suspended fibroblasts
Session: Imaging technics and applications I Chair: Prof. Dr. Jörg Langowski
11:00 - 11:30 Prof. Dr. Alexandra Ros, ASU Tempe, Arizona, USA (Invited Talk)
Deterministic Absolute Negative Mobility for Sub-Micrometer Particle and Organelle Separation
11:30 - 11:50 Christoph Spahn, Frankfurt, Germany (Student Award)
Sequential localization microscopy of different molecule classes in Escherichia coli
11:50 - 12:10 Franziska Fricke, Frankfurt, Germany (Student Award)
Counting membrane proteins with single-molecule localization microscopy
12:10 - 12:30 Narain Karedla, Göttingen, Germany (Student Award)
Simultaneous measurement of the three-dimensional orientation of excitation and emission
dipoles

6
Saturday 10.10.2015
Session: Imaging technics and applications II Chair: Prof. Dr. Alexandra Ros
9:00 - 9:30 Prof. Dr. Jörg Enderlein, Göttingen, Germany (Invited Talk)
Image Scanning Microscopy (ISM)
9:30 - 9:50 Dr. Andreas Neef, Göttingen, Germany
The biophysical basis of the high-bandwidth information encoding in cortical neurons
9:50 - 10:10 Dr. Rys Dowler, PicoQuant GmbH, Berlin, Germany
Advanced Pulse Pattern Generation and Fine Tuning for STED Microscopy
10:10 - 10:30 Dr. Martin Pauli, Würzburg, Germany
Elucidating the molecular architecture of endplate active zones
Session: Localization microscopy Chair: Prof. Dr. Jörg Enderlein
11:00 - 11:30 Prof. Dr. Markus Sauer, Würzburg, Germany (Invited Talk)
Frontiers in single-molecule based super-resolution microscopy
11:30 - 11:50 Dr. Mila Paul, Würzburg, Germany
Molecular architecture of parallel fibre-to-purkinje cell active zones elucidated by dSTORM
11:50 - 12:10 Dr. Sven Proppert, Würzburg, Germany
12:10 - 12:30 Dr. Pablo Mateos-Gil, Würzburg, Germany
Super-resolution imaging of plasma membrane proteins with click chemistry
12:10 - 12:30 AWARDS AND CLOSING REMARKS

7
Abstracts oral presentations
(Listed alphabetically by last name)

8
Single-molecule studies provide new insights into MET receptor activation
Marina S. Dietz1, Franziska Fricke1, Daniel Haße2, Hartmut H. Niemann2, Mike Heilemann1
1Institute of Physical and Theoretical Chemistry, Johann Wolfgang Goethe-University, Frankfurt
(Main), Germany
2Department of Chemistry, Bielefeld University, Bielefeld, Germany
The human receptor tyrosine kinase MET is involved in vertebrate development and plays an
important role during tissue regeneration [1]. Furthermore, it was found that MET is the target of
different infectious bacteria, amongst them Listeria monocytogenes that induces bacterial uptake
through the surface protein internalin B (InlB) and causes human listeriosis.
As many details of MET activation are still unclear, we study activation and association of MET
using single-molecule fluorescence microscopy [2, 3]. In particular, we use single-molecule
localization microscopy, single-molecule photobleaching and single-particle tracking to elucidate
the mechanism of receptor activation. Our studies reveal high-affinity binding of the bacterial
ligand InlB to the MET receptor and partially preformed MET dimers with an increase in receptor
dimers upon InlB binding [4, 5].
[1] Birchmeier C, Birchmeier W, Gherardi E, Vande Woude GF (2003) Met, metastasis, motility and more. Molecular Cell Biology 4, 915.
[2] Heilemann M, van de Linde S, Mukherjee A, Sauer M (2009) Super-resolution imaging with small organic fluorophores. Angewandte Chemie
48, 6903.
[3] Fricke F, Dietz MS, Heilemann M (2015) Single-molecule methods to study membrane receptor oligomerization. ChemPhysChem 16, 713.
[4] Dietz MS, Fricke F, Krüger CL, Niemann HH, Heilemann M (2014) Receptor–Ligand Interactions: Binding Affinities Studied by Single-Molecule
and Super-Resolution Microscopy on Intact Cells. ChemPhysChem 15, 671.
[5] Dietz MS, Haße D, Ferraris DM, Göhler A, Niemann HH, Heilemann M (2013) Single-molecule photobleaching reveals increased MET receptor
dimerization upon ligand binding in intact cells. BMC Biophysics 6, 6.

9
Advanced Pulse Pattern Generation and Fine Tuning for STED Microscopy
Rhys Dowler, Marcelle König, Paja Reisch, Alexander Glatz, Sebastian Tannert, Thomas Schönau,
Romano Härtel, Tino Röhlicke, Marcus Sackrow, Matthias Patting, Felix Koberling, Rainer
Erdmann
PicoQuant GmbH, Berlin, Germany
Stimulated Emission Depletion (STED) microscopy has evolved into a well-established method
offering optical superresolution below 50 nm. Optimal optical resolution can be achieved through
running both excitation and depletion lasers in picosecond pulsed mode as well as fully exploiting
the photon arrival time information using time-resolved single photon counting (TCSPC). Non-
superresolved contributions can be easily dismissed through timegated detection or a more
detailed fluorescence decay analysis. Furthermore, these two methods allow for accurate
separation of different fluorescent species. We present here a new generation of our VisIR 765
”STED” depletion laser, featuring a pulse length and beam shape optimized for STED microscopy.
The temporal overlap between excitation and STED laser pulses can be adapted specifically to
different fluorescence lifetimes thanks to our fully computer controlled multichannel delay
generator SOM-D. The SOM-D allows for the easy introduction of electronic delays between laser
channels with time resolutions below 50 ps. Interleaved pulse patterns can also be realized with
the SOM-D, allowing the illumination to be cycled between STED and non-STED excitation during
a measurement. Such interleaved excitation patterns are advantageous in the identification and
elimination of unwanted STED induced processes on the nanosecond timescale. Examples from
blinking and photobleaching in single molecule imaging as well as in fluorescence correlation
spectroscopy (STED-FCS) will be given. This extended STED functionality along with improved data
throughput are the latest extensions to the confocal microscope platform MicroTime 200 and will
soon be available as upgrade for existing systems.

10
Image Scanning Microscopy (ISM)
Jörg Enderlein
III. Institute of Physics – Biophysics, Georg-August-University Göttingen
Friedrich-Hund-Platz 1, D-37077 Göttingen, Germany
Classical fluorescence microscopy is limited in resolution by the wavelength of light (diffraction
limit) restricting lateral resolution to ca. 200 nm, and axial resolution to ca. 500 nm (at typical
excitation and emission wavelengths around 500 nm). However, recent years have seen a
tremendous development in high- and super-resolution techniques of fluorescence microscopy,
pushing spatial resolution to its diffraction-dictated limits and much beyond, and culminating in
the Nobel Prize for Chemistry in 2014. I will present a short introduction into the subject and will
then focus on one new and clever method which extends the resolution and contrast of imaging
for nearly no extra cost: Image Scanning Microscopy and all its related techniques.
Image Scanning Microscopy (ISM) is a recently developed technique which is equivalent and
alternative to the better known Structured Illumination Microscopy (SIM). In ISM, the focus of a
conventional laser-scanning confocal microscope (LCSM) is scanned over the sample, but instead
of recording only the total fluorescence intensity for each scan position, as done in conventional
operation of an LCSM, one records a small image of the illuminated region. The result is a four-
dimensional stack of data: two dimensions refer to the lateral scan position, and two dimensions
to the pixel position on the chip of the image-recording camera. This set of data can then be used
to obtain a super-resolved image with doubled resolution, analogously to what is achieved with
SIM. However, ISM is conceptually and technically much simpler, suffers less from sample
imperfections like refractive index variations, and can easily be implemented into any existing
LSCM. Since its first experimental realization in 2010 by our group, several research groups
around the world have adopted the idea and have developed clever and powerful variations. I
will present these new developments and the principal ideas behind them.

11
Franziska Fricke1, Joel Beaudouin2, Sjoerd van Wijk3, Ivan Dikic4, Roland Eils2, Mike Heilemann1
1Institute of Physical and Theoretical Chemistry, Goethe University, Frankfurt, Germany
2Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg,
Germany
3Institute for Experimental Cancer Research in Pediatrics, Goethe University, Frankfurt,
Germany
4Institute of Biochemistry II, Goethe University Medical School, Frankfurt, Germany
Single-molecule localization microscopy (SMLM) has emerged as a key super-resolution imaging
technique [1]. It relies on bright photoswitchable fluorescent probes including fluorescent
proteins and organic dyes that are activated in sparse numbers over time and localized with high
precision. Coordinates of individual fluorophores construct the super-resolved image but can also
be exploited for quantitative evaluation including spatial distribution analysis and molecular
counting. In recent years, quantitative SMLM was shown to be particularly useful for studying the
stoichiometry of membrane proteins in intact cells [2]. In contrast to single-molecule
photobleaching approaches, it is not limited to low protein surface densities owing to the super-
resolving nature of the method. Nevertheless, reliable protein counting with SMLM is often
hampered: The blinking behavior of photoswitchable probes can introduce overcounting
artifacts. This can be overcome by introducing an artificial dark time that combines fluorophore
emissions closely clustered in time. Undercounting can however occur when not every target is
fluorescently tagged or blinking cycles of fluorophores overlap in time. Fluorescent proteins (FPs)
are attractive probes for quantitative SMLM because they allow stoichiometric labeling of the
target structure. We report on a simple strategy for probing the stoichiometry of membrane
proteins using photoswitchable FPs. It is based on a kinetic model [3] and does not depend on an
artificial dark time for reliable quantification. After validating our approach using membrane
proteins with defined stoichiometry, we demonstrate subunit counting of membrane receptors
and viral glycoprotein in intact cells.
[1] M Sauer, J Cell Sci 126, 3505-3513 (2013).
[2] F. Fricke et al., ChemPhysChem (2014).
[3] S.-H. Lee et al., Proc. Natl. Acad. Sci. 109, 17436-17441 (2012).

12
Mechanosensitive channels in bacteria gated by a mechanical load
R. Garces and C.F. Schmidt
The regulation of growth and integrity of bacteria is critically linked to mechanical stress. For
instance, to carry out the metabolic tasks, bacteria must keep a high difference of pressure
(turgor) with respect to the environment. This pressure difference (in the order of 1 atm) is
supported by the cell envelope, in brief, a composite of lipid membranes and a rigid cell wall.
Turgor is controlled by the ratio of osmolytes inside and outside bacteria and thus, can experience
abrupt increases upon osmotic downshock. The structural integrity of bacteria relies in the
mechanical response of the cell wall and in the action of mechanosensitive (MS) channels,
membrane proteins that release solutes in response to tension in the cell envelope [1]. There are
two main families of MS channels, MscS and MscL. The respective parameters of the gating have
been studied in spheroplast, via patch clamp technique [2][3]. However a precise characterization
of their action in the ‘complete bacteria’ is still lacking. We present an study of MS channels
activated by the stress associated to a mechanical force exerted by the tip of an Atomic force
microscopy (AFM) when a bacterium E.coli is indented "in vivo".
[1] Ian. R. Booth et al. Mechanosensitive channels in bacteria: signs of closure? Nature Reviews Microbiology 5, 431-440 (June 2007)
[2] Natalia Levina. et al. Protection of Escherichia coli cells against extreme turgor by activation of MscS and MscL mechanosensitive channels:
identification of genes required for MscS activity. EMBO J. 18, 1730-1737 (1999)
[3] Sergei Sukharev et al. The gating mechanism of the large mechanosensitive channel MscL. Nature 409, 720-724 (8 February 2001)

13
Simultaneous measurement of the three-dimensional orientation of excitation
and emission dipoles
Narain Karedla, Simon C. Stein, Dirk Hähnel, Ingo Gregor, Anna Chizhik, Jörg Enderlein
The emission properties of most fluorescent emitters, such as organic dye molecules or solid-
state color centers, are well described by an oscillating electric dipole model. However, the
orientations of their excitation and emission dipoles are, in most cases, not parallel. Although
single-molecule excitation and emission dipole orientation measurements have been performed
in the past, no experimental method has so far looked at the three-dimensional excitation end
emission dipole geometry of individual emitters simultaneously. We present the first
experimental study, by combining radially-polarized laser scanning together with defocused
imaging, to measure both the excitation and emission dipole orientations for single molecules,
which allows us to sample the distribution of their mutual orientation. We find an unexpectedly
broad distribution of the angle between both dipoles which we attribute to the interaction
between the observed molecules and the substrate they are immobilized on. Further, this method
is ideal for investigating the dimensionality and orientations of the TDMs of quantum dots, which
are dramatically modified in the vicinity of metal nanostructures.

14
Chromatin dynamics are controlled by nuclear lamin A
1, 2Giulia Marcarini, 1, 3Jan W. Krieger, 2Giuseppe Chirico, 1, 3Jörg Langowski
1Division Biophysics of Macromolecules, DKFZ, INF 580, D-69120 Heidelberg, Germany
2Department of Physics, University Milano-Bicocca, Italy
3Interdisciplinary Center for Scientific Computation (IWR), University of Heidelberg, INF 368, D-
69120 Heidelberg, Germany
Important biological processes such as gene activity, replication, transcription and recombination
depend critically on the organization and dynamics of the genome. In particular, the viscoelastic
properties of the cell nucleus and their connection with gene function have become a focus of
interest recently [1]. Microscopy techniques that allow the visualization of chromatin dynamics
on fast time scales, and the dynamics of proteins in the chromatin network, are therefore valuable
tools for understanding the mechanism of genome function. Fluorescence correlation
spectroscopy (FCS) is a typical microscopic technique for characterizing intracellular protein
mobility, which offers fast time resolution but so far has been limited to single-point
measurements in the focus of a laser beam. Although we have collected protein mobility maps
by point-to-point FCS [2], this method is extremely time-consuming and not very feasible for live
cell measurements. SPIM-FCS is a new method that combines the speed of FCS with the possibility
of acquiring mobility data on an entire two-dimensional cross-sections of cells [3], providing
diffusion coefficients, flow velocities and concentrations in an imaging mode. Two-color
fluorescence cross-correlation spectroscopy (SPIM-FCCS) also allows one to image molecular
interactions [4]. Here we present new data on the dynamics of interphase chromatin, measured
by FCS analysis of fast image series from light sheet microscopy of fluorescently labeled histones
in interphase HeLa cell nuclei. We show that the random motion of the chromatin network is
subdiffusive, that is, the effective diffusion coefficient decreases for slow time scales. In a lamin
A knockout cell line, on the other hand, the diffusion changes to normal. While a similar effect
has been observed by single particle tracking of telomers [1], here we have established that this
is a property of the entire chromatin network. Finally, we present first data on the correlated
motion of lamin A and histones measured by imaging-FCCS. Our conclusion is that lamin A plays
a central role for determining the elasticity of the chromatin network and to help maintaining
local ordering of interphase chromosomes.
[1] I. Bronstein, Y. Israel, E. Kepten, S. Mai, Y. Shav-Tal, E. Barkai, Y. Garini, Phys Rev Lett 2009, 103, 018102.
[2] N. Dross, C. Spriet, M. Zwerger, G. Muller, W. Waldeck, J. Langowski, PLoS One 2009, 4, e5041.
[3] T. Wohland, X. Shi, J. Sankaran, E. H. Stelzer, Opt Express 2010, 18, 10627-10641.
[4] J. W. Krieger, A. P. Singh, C. S. Garbe, T. Wohland, J. Langowski, Opt Express 2014, 22, 2358-2375.

15
Super-resolution imaging of plasma membrane proteins with click chemistry
Pablo Mateos Gil
Department of Biotechnology and Biophysics, Biocenter, Julius-Maximilian-University Würzburg,
Würzburg, Germany
Besides its function as a passive cell wall, plasma membrane (PM) serves as a platform for
different physiological processes such as signal transduction and cell adhesion, determining the
ability of cells to communicate with the exterior and form tissues. Therefore, the molecular
distribution of PM components at the nanoscale has potential implications in several biological
fields such as cell development, neurobiology, or immunology. Fluorescence imaging of
membrane components at the nanoscale was limited in the past due to the resolution barrier
imposed by the diffraction of light. This problem has been recently overcome by super-resolution
fluorescence microscopy methods which have revealed the presence of nanoclusters of specific
proteins in the cell membrane [1, 2]. However, probing the existence of universal mechanisms
underlying mesoscale spatial distribution of all PM membrane proteins remains challenging [3].
To test this hypothesis, more global approaches aimed to image simultaneously a large
population of membrane proteins is required. Here I will present a bioorthogonal chemical
strategy, based on click chemistry and metabolic labeling [4], to study putative plasma membrane
organization, using direct STochastic Optical Reconstruction Microscopy (dSTORM) to visualize
newly synthesized proteins containing cotranslational incorporated non-canonical clickable
amino acids.
[1] Dani, A.; Huang, B.; Bergan, J.; Dulac, C. and Zhuang, X. (2010). Superresolution imaging of chemical synapses in the brain., Neuron 68 : 843-
856.
[2] Bar-On, D.; Wolter, S.; van de Linde, S.; Heilemann, M.; Nudelman, G.; Nachliel, E.; Gutman, M.; Sauer, M. and Ashery, U. (2012). Super-
resolution imaging reveals the internal architecture of nano-sized syntaxin clusters., J Biol Chem 287 : 27158-27167.
[3] Kusumi, A.; Fujiwara, T. K.; Chadda, R.; Xie, M.; Tsunoyama, T. A.; Kalay, Z.; Kasai, R. S. and Suzuki, K. G. N. (2012). Dynamic organizing inciples
of the plasma membrane that regulate signal transduction: commemorating the fortieth anniversary of Singer and Nicolson's fluid-mosaic
model., Annu Rev Cell Dev Biol 28 : 215-250.
[4] Beatty, K. E.; Liu, J. C.; Xie, F.; Dieterich, D. C.; Schuman, E. M.; Wang, Q. and Tirrell, D. A. (2006). Fluorescence visualization of newly
synthesized proteins in mammalian cells., Angew Chem Int Ed Engl 45 : 7364-7367.

16
The biophysical basis of the high-bandwidth information encoding in cortical
neurons
1,2Andreas Neef
1Max Planck Institute for Dynamics and Self-Organization
2BCCN group ‘Biophysics of neural information encoding’, Göttingen, Germany
A cerebral neuron receives a continuously fluctuating input through thousands of synapses. It
encodes and relays this input to thousands of downstream neurons using action potentials.
Therefore, the dynamics, with which discrete action potentials are generated in response to a
continuous input, represents a fundamental bottleneck for the flow of information in neural
populations. In the last decade it became clear that the bandwidth of information encoding in
neural populations in the cerebral cortex is much higher than previously predicted by simulations
with conductance based models. The biophysical basis of this large bandwidth is not understood,
as even basic parameters such as sodium channel surface density and kinetics are still under
debate. The work presented here uses high resolution electrophysiology and fluorescence
microscopy to quantify sodium channel properties and distributions in neurons and relate them
to the ability to encode information with a high bandwidth.
A characterization of sodium channels properties in the cell bodies of cortical pyramidal cells
allowed us to conclude that each square micrometer contains 20 to 30 sodium channels. By
combining current clamp and immunofluorescence in cultured hippocampal neurons we could
achieve semi-quantitative fluorescence labeling and obtain estimates for the axonal density of
sodium channels.
We found the bandwidth of information encoding to be similar between cultured neurons and
neurons in slices. Using cultured neurons as a model system, we studied the maturation of
neuronal properties in the first weeks in culture. In parallel to the increased bandwidth, other
neuronal properties changed: the axonal sodium channel density, the dendritic morphology and
the sub-micrometer organization of axon initial segment structure. The presentation details, how
those properties influenced the bandwidth of information encoding.

17
Molecular architecture of parallel fibre-to-purkinje cell active zones elucidated
by dSTORM
1Mila M. Paul, 1Sven Proppert, 1Martin Pauli, 2Markus Sauer, 1Manfred Heckmann
1Institute of Physiology, Department of Neurophysiology, Julius-Maximilian-University
Würzburg, Würzburg, Germany
2Department of Biotechnology and Biophysics, Biocenter, Julius-Maximilian-University
Würzburg, Würzburg, Germany
Presynaptic active zones (AZs) are elaborated subneuronal compartments specialized for vesicle
fusion and neurotransmitter release. They show complex protein-protein interactions within a
range of a few hundred nanometers which require the use of high-resolution imaging techniques
to analyze their molecular architecture. Here, we used 2D and 3D direct stochastic optical
reconstruction microscopy (dSTORM, Heilemann et al., 2008; van de Linde et al., 2011) to
investigate the arrangement of parallel fibre-to-purkinje cell AZs in the cerebellum of male 3-4
weeks old C57BL/6 mice. Presynaptic parallel fibre boutons typically harbour one single AZ with
a small number of docked vesicles (Xu-Friedman et al., 2001) in a coupling distance of less than
30 nm to voltage-gated calcium channels (Schmidt et al., 2013). We used parasagittal cryoslices
of 1 μm thickness which allowed us to identify single AZs in side view using an N-terminal antibody
against the scaffolding protein Bassoon (Bsn). Each Bsn cluster had the form of a rectangular
badge-like perforated structure and a length of 0.52 ± 0.09 μm. We imaged presynaptic PQ-type
calcium channels relatively to Bsn and found 3.5 ± 1.9 clusters per AZ. With an antibody against
the N-terminal, vesicle-binding region of the AZ-protein RIM (Rab3-interacting molecule) we
identified 4.6 ± 1.6 RIM-clusters per AZ. Correlation between localization numbers of Bsn and RIM
was relatively high (r = 0.5 with p < 0.001) and lower between Bsn and calcium channels (r = 0.28
with p < 0.05). These data illustrate the contribution of superresolution imaging techniques to a
better understanding of the molecular architecture of chemical synapses in the mammalian brain.
Heilemann M, van de Linde S, Schüttpelz M, Kasper R, Seefeldt B, Mukherjee A, Tinnefeld P, Sauer M. (2008) Subdiffraction-resolution
luorescence imaging with conventional fluorescent probes. Angew Chem Int Ed Engl 47:6172-6.
Schmidt H, Brachtendorf S, Arendt O, Hallermann S, Ishiyama S, Bornschein G, Gall D, Schiffmann SN, Heckmann M, Eilers J (2013) Nanodomain
coupling at an excitatory cortical synapse. Curr Biol. 23:244-9.
van de Linde S, Löschberger A, Klein T, Heidbreder M, Wolter S, Heilemann M, Sauer M (2011) Direct stochastic optical reconstruction
microscopy with standard fluorescent probes. Nat. Protocols 6: 991-1009.
Xu-Friedman MA, Harris KM, Regehr WG (2001) Three-dimensional comparison of ultrastructural characteristics at depressing and facilitating
synapses onto cerebellar Purkinje cells. J Neurosci. 21:6666-72.

18
Elucidating the molecular architecture of endplate Active Zones
1Martin Pauli, 2Markus Sauer, 3Claudia Sommer, 1Manfred Heckmann
1Department of Neurophysiology, Julius-Maximilians-University Würzburg, Germany
2Department of Biotechnology & Biophysics, Julius-Maximilians-Universität Würzburg, Germany
3Department of Neurology, Universitätsklinikum Würzburg, Germany
Active Zones (AZs) are complex meshworks of proteins that organize presynaptic vesicle traffic.
Electron microscopy (EM) of AZs at vertebrate neuromuscular junctions revealed, symmetrical,
bilateral sub-compartments, which are called pegs, ribs and beams (Harlow et al., 2001;
Nagwaney et al., 2009). Pegs, ribs and beams are small, less than 100 nm apart and their
molecular composition is still unclear. To address this we use immunohistochemistry and
dSTORM, which offers a spatial resolution of about 20 nm, (Heilemann et al., 2008; van de Linde
et al. 2011; Ehmann et al., 2014) in semithin Tokayasu cryosections of the levator auris muscle of
8 to 10 week old mice. We stain postsynaptic nicotinic acetylcholinreceptors in perpendicular cut
endplates with Alexa 647-labelled Bungarotoxin and are able to resolve posynaptic junctional
folds that extend over several micrometers. Furthermore we can identify presynaptic AZs vis-à-
vis the postsynaptic folds and determine their spatial distribution and rotational orientation
relative to the longitudinal axis of the postsynaptic fold. We use estimates for the arrangement
of pegs, ribs and beams and AZ-size from EM (Nagwaney et al., 2009) and map epitopes of core
AZ-proteins such as bassoon, piccolo and Clathrin.
Ehmann N, van de Linde S, Alon A, Ljaschenko D, Keung XZ, Holm T, Rings A, DiAntonio A, Hallermann S, Ashery U, Heckmann M, Sauer M, Kittel
RJ (2014) Quantitative superresolution imaging of Bruchpilot distinguishes active zone states. Nat Commun 5:4650.
Harlow ML, Ress D, Stoschek A, Marshall RM, McMahan UJ. (2001) The architecture of active zone material at the frog's neuromuscular junction.
Nature 409:479-84.
Heilemann M, van de Linde S, Schüttpelz M, Kasper R, Seefeldt B, Mukherjee A, Tinnefeld P, Sauer M. (2008) Subdiffraction-resolution
fluorescence imaging with conventional fluorescent probes. Angew Chem Int Ed Engl 47:6172-6.
Nagwaney S, Harlow ML, Jung JH, Szule JA, Ress D, Xu J, Marshall RM, McMahan UJ. (2009) Macromolecular connections of active zone material
to docked synaptic vesicles and presynaptic membrane at neuromuscular junctions of mouse. J Comp Neurol 513:457-68.
van de Linde S, Löschberger A, Klein T, Heidbreder M, Wolter S, Heilemann M, Sauer M (2011) Direct stochastic optical reconstruction
microscopy with standard fluorescent probes. Nat. Protocols 6: 991-1009.

19
3D super-resolution measurements of brain tissue after elimination of
aberrations using standard cubic B-spline interpolation for calibration
1Sven Proppert, 1Martin Pauli, 1Mila Paul, 2Steve Wolter, 3Thorge Holm, 1Manfred Heckmann,
3Markus Sauer
1Department of Neurophysiology, Institute of Physiology, Julius-Maximilian-University
Würzburg, Germany
2Google Inc., München, Germany
3Department of Biotechnology and Biophysics, Biocenter, Julius-Maximilian-University
Würzburg, Germany
Three-dimensional super-resolution imaging became widely used in the single-molecule
localization community. Today, a variety of algorithms to calibrate these measurements are in
use and some of which even account for imperfections in the optical pathway. However, our
investigations led to the conclusion that the most popular algorithms strongly rely on user-input
which renders them little suited for everyday use. We therefore present, that standard cubic B-
splines, as commonly used in interpolation problems, are very robust in use without any loss in
precision or accuracy. They are thus ideally suited for beginners and for investigators that need
the flexibility to deal, for example, with aberrations.
Furthermore, sources of aberrations commonly encountered in super-resolution microscopy will
be recapitulated and their impact will be demonstrated on slices of brain tissue taken from mice.
It will be shown, that measuring in the presence of a refractive index mismatch can significantly
disturb the results while aberration-free imaging is possible even in comparatively thick slices
when objectives with a matching immersion medium are used.
To cut it short, we will describe how 3D calibrations can be performed mostly fail-safe on a daily
basis in the aberration-free case and even in the presence of refractive index induced aberrations.
With our microscope calibrated accordingly, we are able to achieve resolutions of about 20
nanometer laterally and 60 nanometer axially over an axial range of one micrometer in adherent
cells and, more importantly, as well in tissue slices with a thickness of more than ten micrometer.
We also demonstrate that the microscope is capable of precise and accurate two-color-3D
imaging over an axial range of 100 micrometer.

20
Cell-Matrix-Mechanics Dictates Stem Cell Fate via Cytoskeleton Structure
Florian Rehfeldt
III. Institute of Physics – Biophysics, Georg-August-University Göttingen Friedrich-Hund-Platz 1,
D-37077 Göttingen, Germany
The mechanical properties of microenvironments in our body vary over a broad range and are as
important to cells as traditional biochemical cues. An especially striking experiment of this
mechano-sensitivity demonstrated that systematic variation of the Young’s elastic modulus E of
the substrate can direct the lineage differentiation of human mesenchymal stem cells (hMSCs).
To elucidate the complex interplay of physical and biochemical mechanisms of cellular mechano-
sensing, well-defined extracellular matrix (ECM) models are essential. While elastic substrates
made of poly-acrylamide (PA) are widely in use, they have the potential drawback that the
precursors are cytotoxic and therefore do not allow for 3D culture systems. Here, a novel
biomimetic ECM model based on hyaluronic acid (HA) was successfully established that exhibits
a widely tuneable and well-defined elasticity E, enables 2D and 3D cell culture and enables us to
mimic a variety of distinct in vivo microenvironments. Quantitative analysis of the structure of
acto-myosin fibers of hMSCs on elastic substrates with an order parameter S, reveals that the
stress fiber morphology is an early morphological marker of mechano-guided differentiation and
can be understood using a classical mechanics model. Furthermore, the cytoskeleton also dictates
the shape of the nucleus and lends support to a direct mechanical matrix-myosin-nucleus
pathway.

21
Deterministic Absolute Negative Mobility for Sub-Micrometer Particle and
Organelle Separation
1, 2Jinghui Luo, 3Katherine Muratore, 3, 4Edgar Arriaga, 1, 2 Alexandra Ros
1School of Molecular Sciences, Arizona State University, Tempe, Arizona, USA.
2Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University,
Tempe, Arizona, USA
3Department of Chemistry, University of Minnesota, Minneapolis, Minnesota, USA.
4Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota,
Minneapolis, Minnesota, USA
The heterogeneity of organelles related to subpopulations in size represents an important yet
little explored factor determining disease initiation and progression. For example, mitochondria
are known to exists in ‘normal’ sizes around 300 nm as well as giant mitochondria of sizes >1μm,
which have been observed in various pathological states and models of aging. Current analytical
approaches are however not suitable to fractionate mitochondria by size. Here, we propose to
exploit a counter-intuitive, yet efficient migration mechanism for μm- and sub-μm particles,
taking advantage of particle transport in a nonlinear post array in a microfluidic device under the
periodic action of electrokinetic and dielectrophoretic forces. Regimes were revealed in which
particle migration opposite to the average applied force occurs for larger particles – a typical
signature of absolute negative mobility (ANM) – whereas normal response is obtained for smaller
particles. The combination with dielectrophoresis induces a deterministic migration component
which allows the application for sub-μm sized (bio)particles. Deterministic ANM (dANM) was
characterized in numerical modeling revealing optimized driving parameters for μm-sized beads.
Furthermore, we experimentally prove dANM with colloidal particles in excellent agreement with
the employed numerical model. The observed dANM is characterized by improved migration
speed at least two orders of magnitude higher compared to previous ANM systems with colloids.
In addition, we were able to induce dANM for mouse liver mitochondria representing the first
demonstration of ANM migration with biological species. We envision that the efficient size
selectivity can be advantageously employed in organelle sub-population studies.

22
Physical Properties of Fibrinogen Matrices Control the Adhesion of Blood Cells
Prof. Dr. Robert Ross
Arizona State University, Department of Physics Tempe, Arizona 85287-1504
Adsorption of fibrinogen on fibrin clots and other surfaces strongly reduces integrin-mediated
adhesion of platelets and leukocytes with implications for the surface-mediated control of
thrombus growth and blood compatibility of biomaterials. For example, adsorption of fibrinogen
on the luminal surface of biomaterials is a critical early event during the interaction of blood with
implanted vascular graft prostheses which determines their thrombogenicity. In my talk, I will
present the application of atomic force microscopy based force spectroscopy techniques
combined with optical microscopy to quantify the adhesive properties of fibrinogen matrixes on
the single cell level and to study the mechanism behind the transformation of highly adhesive to
non-adhesive surfaces. These results provide improved understanding of the molecular events
underlying thrombogenicity of vascular grafts.

23
Frontiers in single-molecule based super-resolution microscopy
Markus Sauer
Department of Biotechnology & Biophysics, Julius Maximilian University Würzburg, Am Hubland,
97074 Würzburg, Germany
Single-molecule based super-resolution microscopy (localization microscopy) provides the ability
to unravel the structural organization of cells and the composition of biomolecular assemblies at
a spatial resolution well below the diffraction limit approaching virtually molecular resolution.
Constant improvements in fluorescent probes, efficient and specific labeling techniques as well
as refined data analysis and interpretation strategies further improved the power of super-
resolution microscopy. Today, it allows us to interrogate how the distribution and stoichiometry
of interacting proteins in subcellular compartments and molecular machines accomplishes
complex interconnected cellular processes. However, despite the relative simplicity of the
microscope setups and the availability of commercial instruments, localization microscopy faces
unique challenges. While achieving super-resolution is no longer a problem, the question we have
to ask ourselves is whether localization microscopy images can be ‘trusted’ to reveal novel
biological insights. Furthermore, super-resolution microscopy requires high irradiation intensities
to use the limited photon budget efficiently and such high photon densities are likely to induce
cellular damage in live-cell experiments.
Here, we discuss how super-resolution microscopy artifacts, such as non-existing membrane
clusters are generated by inappropriately set experimental conditions and inexperience. In
addition, we demonstrate the influence of irradiation intensity, illumination-mode, wavelength,
light-dose, temperature and fluorescence labeling on the survival probability of different cell
lines.

24
Force fluctuations in three-dimensional suspended fibroblasts
Florian Schlosser, Christoph F. Schmidt, Florian Rehfeldt
Cells are sensitive to mechanical cues from their environment and at the same time generate and
transmit forces to probe and to adapt to their surroundings. Key players in the generation of
contractile forces are acto-myosin structures. To test forces and elasticity of cells not attached to
a substrate, we used a dual optical trap to suspend 3T3 fibroblasts between two fibronectin-
coated beads. We analyzed the correlated motions of the beads with high bandwidth. A
combination of active and passive microrheology allowed us to measure the non-equilibrium
force fluctuations as well as the elastic properties of the cell. We found that cortical forces deform
the cell from its round shape in the frequency regime from 0.1 to 10 Hz. Biochemical perturbation
experiments using blebbistatin for myosin inhibition and nocodazole for microtubule
depolymerization show that cell stiffness and cortical force fluctuations highly depend on acto-
myosin activity but not on microtubules. Serum-starvation also largely reduced the fluctuation
amplitude. A force-clamp allowed us to observe cells under defined constant forces. Combining
our optical trap with a confocal microscope allowed us to image the three-dimensional actin
distribution of Life-Act transfected cells during the force measurements.

25
Active Matter in Cells
Christoph F. Schmidt
Living cells are out of thermodynamic equilibrium, most importantly due to metabolism driving
chemical non-equilibrium. Some of the energy dissipated is used by cells to create forces and
motion. I will focus on non-equilibrium dynamics in the actin cell cytoskeleton. Actin networks
inside living cells adopt different architectures to perform specific cellular processes such as cell
division, motility and generation of cell polarity. I will give an overview of recent projects and will
focus on a model for the actin cortex. We reconstituted actomyosin cortices using Xenopus egg
extract in oil emulsions. Using single-walled carbon nanotubes as non-perturbing probes, we
performed high-resolution mapping of cortical fluctuations in these model systems. We found
non-equilibrium transitions between distinct steady states when connectivity was tuned by cross-
linking. We have developed a model to explain the observed dynamic steady states in which
connectivity percolation enables long-range stress propagation by myosin motors which in turn
leads to the re-distribution of the network and, in turn, structured arrangements of motors.

26
Sequential localization microscopy of different molecule classes in Escherichia
coli
1Christoph Spahn, 2Ulrike Endesfelder, 1Mike Heilemann
1Single Molecule Biophysics, Institute of Physical and Theoretical Chemistry, Goethe-University
Frankfurt, 60438 Frankfurt, Germany
2Max Planck Institute for Terrestrial Microbiology and LOEWE Center for Synthetic Microbiology
(SYNMIKRO), Department of Systems and Synthetic Microbiology,Marburg, Germany
Single-molecule localization microscopy (SMLM) is a new methodological toolbox which allows
addressing questions on organization, interaction and dynamics of individual proteins in a cellular
context: Single-molecule registration allows for quantitative molecular counting and for single-
molecule tracking, as well as for discovering sub-populations and heterogeneities which are
otherwise hidden through ensemble averaging. This provides, next to super-resolution images,
quantitative, single-molecule resolved information (1). We here demonstrate easily applicable
protocols for SMLM and correlative imaging of RNA polymerase distributions and dynamics,
membrane and chromosome structure in Escherichia coli (2-4). These strategies are based on
sequential imaging approaches bacterial cells that are embedded in a porous collagen matrix after
fixation and facilitate quantitative PALM imaging of a protein of interest, followed by PAINT
imaging of the conserved membrane using the lipophilic dyes Nile Red and R6G. The embedded
bacterial cells can be subsequently membrane-permeabilized and click labeled using the
thymidine analogue 5’-ethynyl-2’-deoxyuridine (EdU), which was incorporated into nascent DNA
during growth prior to fixation. The registration of all three channels thus allows to investigate
the nucleoid ultrastructure, its positioning within the highly resolved cell cylinder and the spatial
relationship of the protein of interest, in example the RNA polymerase, and the bacterial
chromosome, We show that during fast growth, RNA polymerase is associated to the nucleoid
throughout the whole cell cycle and mainly populates the nucleoid surface, while it cannot
penetrate the condensed chromosome core. This structural observation was further validated
when correlating trajectories of single RNA polymerases to the nucleoid in a fast on-the-slide
fixation and post-labeling approach. Overall, our results show that it is strikingly interesting to
apply similar protocols e.g. to other nucleoid-associated proteins in order to draw a general
picture about chromosome architecture and organization in various growth conditions at such a
small scale.
(1) Endesfelder and Heilemann (2014), Nature Methods, 11, 235-238.
(2) Endesfelder et al. (2013), Biophys Journal, 105(1), 172-181.
(3) Spahn et al. (2014), Journal of Structural Biology, 185 (3), 243-249.
(4) Spahn et al. (2015), Methods Appl. Fluoresc. 3, 014005.

27
Single molecule fluorescence studies of nucleosome dynamics
1Kathrin Tegeler, 1Alexander Gansen, 1Johanna Mehl, 1Katalin Toth, 2Koby Levy, 1Jörg
Langowski
1Abteilung Biophysik der Makromoleküle, Deutsches Krebsforschungszentrum, Heidelberg,
Germany;
2Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel
The nucleosome, as the basic packing unit of chromatin, regulates DNA accessibility and has significant
influence on gene expression. Two copies of each histone protein (H2A, H2B, H3 and H4) build up the
protein octamer, around which approximately 150 bp of DNA are wrapped in 1.75 turns.
The N-terminal tails of the histone proteins protrude from the nucleosome; they are important for inter-
and intranucleosomal interactions. Nucleosome disassembly may be forced in vitro by increasing salt
concentration and may be followed by Förster resonance energy transfer (FRET) between fluorophores on
the DNA. With this technique it was shown that the H3 tail is involved in intranucleosomal interactions by
restricting the DNA breathing motion as well as in the compaction of the nucleosome[1]. We use
reconstituted Xenopus laevis nucleosomes, fluorescently labeled on different positions within the histone
core and along the DNA[2]. This way we can compare the disassembly of nucleosome containing mutated
histones or modifications to the wild type.
Here we focus on the dynamics of the N-tail of X. laevis H3. Mutated versions of H3 (K4C, C110A; K9C,
C110A (K. Luger, CSU, Fort Collins)) labeled with Alexa488 were used to examine whether there are
interactions between the labeled histone H3 tail and the labeled DNA or other labeled histones. First
results show an interaction of the H3 tail and the DNA in vicinity of the dyad axis.
The second part of the project is about the functional relevance of a particular region of the histone H2A.
Molecular dynamic simulations on ‘tailless’ variants of H3 and H2A have suggested conformational
changes affecting two arginines of H2A: a shift of interactions of H2A R81 to an interaction with the DNA,
and of H2A R88 with H3 E105. Furthermore a conformational change of the protruding part of the DNA
was described, which is due to internal conformational changes of histone H2A[3].
From those observations the question arises whether these amino acids are necessary for the
reconstitution of histone octamers and/or nucleosomes, and how do these mutations influence DNA
breathing, unwrapping, and stability of entire nucleosomes. To address these questions we generated
recombinant H2A proteins incorporating site-specific mutations (R81A, R88A, R81AR88A, R81E, R88E,
R81ER88E). The exchange of Arg to Ala leads to a removal loss of the associated positive charge, and in
case of Arg to Glu, the positive charge is replaced by a negative charge. Our results show a decreasing
stability associated with the amino acid position (Wt > R88 > R81 > R81R88).
Combining both projects will help us understand the role of the H3 tail in the overall stability of the
nucleosome, its influence on the amino acids R81 and R88 of H2A and vice versa.
[1] Nurse, N. P., Jimenez-Useche, I., Smith, I. T., & Yuan, C. (2013). Clipping of Flexible Tails of Histones H3 and H4 Affects the Structure and Dynamics of the Nucleosome. Biophysical Journal, 104(5), 1081–1088. doi:10.1016/j.bpj.2013.01.019
[2] Böhm, V., Hieb, A. R., Andrews, A. J., Gansen, A., Rocker, A., Tóth, K., … Langowski, J. (2011). Nucleosome accessibility governed by the dimer/tetramer interface. Nucleic Acids Research, 39(8), 3093–3102. doi:10.1093/nar/gkq1279
[3] Biswas, M., Voltz, K., Smith, J. C., & Langowski, J. (2011). Role of Histone Tails in Structural Stability of the Nucleosome. PLoS Computational Biology, 7(12), e1002279. doi:10.1371/journal.pcbi.1002279

28
COLORFUL NUCLEOSOMES
Katalin Tóth, Alexander Gansen, Yoriko Lill, Jörg Langowski
Biophysics of Macromolecules, DKFZ, Heidelberg, Germany
Beyond genome packaging, histones play an active role in regulating gene activity through post-
translational modifications and sequence variations. Salt-induced disassembly of
mononucleosomes is affected by such small chemical modifications and can be used as a tool to
discover mechanisms of nucleosome opening. We use Förster resonance energy transfer (FRET)
between different parts of the mononucleosome to study these mechanisms. FRET of single
molecules can characterize intermediate states in nucleosome disassembly.
The effect of acetylation of different histones is not cumulative, sometimes even counteracting.
At physiological salt concentrations, DNA ends open upon H3 acetylation and close upon H4
acetylation. At higher salt concentrations both lead to enhanced DNA unwrapping. The release of
H2A-H2B dimers is enhanced by H3 acetylation, H4 acetylation counteracts this process.
Local modifications in prominent positions of the histone tails has high biological impact. Contrary
to acetylation of the entire H4 histone, we found that acetylation of a single lysine (K16) in H4
destabilizes nucleosomes. Replacing H4K16 with glutamine - mimicking acetylation - or the
mutation of other lysines in the H4 tail into glutamine has no effect on the nucleosome stability.
Thus, we suppose that the effect of acetylation is more than just electrostatics.
To probe nonelectrostatic interactions we followed nucleosome disassembly using different
anions. The nucleosome-destabilizing effect significantly decreases in the order NaClO4 > NaCl >
Na-acetate > NaF, according to the Hofmeister series.
The opening pathway suggested by our FRET results seems to be independent of DNA sequence
or methylation, of histone origin, modifications or mutations or of the anions investigated.

29
Abstracts poster presentations
(Listed alphabetically by last name)

30
Correlated calcium and super-resolution imaging for structure-function analysis
of the postsynapse
1Carolin Böger, 2Cyril Hanus, 3Ulrike Endesfelder, 2Erin M. Schuman, 1Mike Heilemann
1Institute for Physical and Theoretical Chemistry, Goethe University Frankfurt, Frankfurt am
Main, Germany
2Max-Planck-Institute for Brain Research, Frankfurt am Main, Germany
3Max-Planck-Institute for Terrestrial Microbiology, Marburg, Germany
During synaptic plasticity, individual excitatory synapses show structural and functional changes
[1]. While at present, synaptic protein copy numbers are available for an average synapse,
spatially dependent changes of synaptic properties have been observed across multiple scales
within individual neurons [2]. So far, the degree of correlation between synaptic activity and
protein organization (localization and quantification) of individual synapses is not clear as well as
how this link varies for neighboring synapses along dendrites within single neurons.
We combine calcium imaging and subsequent direct stochastic optical reconstruction microscopy
[3] to correlate the history of synaptic activity and the corresponding molecular nano-
organization at the level of single synapses and compare synapses with respect to their spatial
relation within individual neurons.
Here, we are focusing on the highly regulated AMPA receptor (i.e. the subunit GluA2) and present
super-resolved images of the synaptic GluA2 composition and synaptic activity visualized with
GCaMP6 in living neurons.
[1] Choquet D, Triller A (2013) The Dynamic Synapse, Neuron 80, 691.
[2] Makino H, Malinow R (2011) Compartmentalized versus global synaptic plasticity on dendrites controlled by experience, Neuron 72, 1001
[3] Heilemann M, van de Linde S, Mukherjee A, Sauer M (2009) Super-resolution imaging with small organic fluorophores. Angewandte Chemie
48, 6903.

31
Comparison of the light-gated ion channels ChR2 and Chronos using Markov-
model driven electro-physiological analysis
1Ulrich Fromme, 1Christoph F. Schmidt, 2, 3Andreas Neef
1 III. Institute of Physics – Biophysics, Georg-August-University Göttingen Friedrich-Hund-Platz 1,
2Bernstein Center for Computational Neuroscience, Göttingen, Germany
3Max Planck Institute for Dynamics and Self-Organization, Göttingen, Germany
The light-gated ion channels channelrhodopsins (ChRs) have become a major tool in experimental
neuro-science due to their low invasiveness and the possibility of genetically targeting specific
cell types. To address specific issues, various ChRs with a wide array of different properties have
been introduced. Here we characterize ‘Chronos’ an especially light sensitive ChR with improved
opening and closing speeds compared to traditional ChRs such as the widely used ChR2. Chronos
shows great promise in various applications, in which good time resolution and low light
intensities are required. We used patch-clamp recordings to characterize the kinetics of the
ensemble of channels, which dictate the performance in applications. From these electro-
physiological measurements we extracted single-molecule properties from model based analysis
and from simulations using newly created Markov models representing the light cycle of these
Channels.

32
Simultaneous measurements of thickness and diffusion of lipid bilayers using
MIET and 2f-FLCS
Sebastian Isbaner, Falk Schneider, Narain Karedla, and Jörg Enderlein
Diffusion plays a key role for passive transport and signaling in cell membranes. A lipid bilayer is
a simple model system for these membranes which has been used extensively for diffusion
studies. As the thickness of a bilayer is just a few nanometers, the diffusion coefficient is usually
an average over both leads. We report here the first simultaneous measurement of thickness and
diffusion coefficients for both leaets of a bilayer by combining metal-induced energy transfer
(MIET) and two focus fluorescence lifetime correlation spectroscopy (2f-FLCS). MIET describes the
phenomenon that a fluorophore is quenched when it is close to a metal surface. Similar to
Foerster resonance energy transfer (FRET), the quenching is distance dependent, and from the
measured lifetime of the fluorophore the distance to the surface is obtained with nanometer
precision. 2f-FLCS uses different fluorescence lifetimes to separate the contribution of spectrally
indistinguishable fluorophores to the intensity correlations. Because of the MIET effect,
fluorophores in the two leaets will have different lifetimes for which 2f-FLCS can determine
individual diffusion coefficients. For a supported lipid bilayer (SLB) prepared by spin coating on an
ITO coated coverslip, we measure a thickness of 6:4 nm. The diffusion constant in the upper leaet
is enhanced by a factor of two, which shows substantial interactions of the lipids with the
substrate.

33
Imaging fluorescence correlation spectroscopy for the investigation of
membrane proteins
Christos Karathanasis, Phoebe H. Young, Marina S. Dietz, Mike Heilemann
Institute of Physical and Theoretical Chemistry, Johann Wolfgang Goethe-University, Frankfurt
(Main), Germany
Membrane receptors are the starting point for signaling cascades in cells [1]. Thus, it is important
to examine their dynamics to gain information on receptor function. Imaging fluorescence
correlation spectroscopy (Imaging FCS) emerged recently as a technique to study membrane
receptors and to create two-dimensional maps of their concentrations and diffusion coefficients
on the cell membrane [2, 3].
This technique was applied to different membrane receptors and membrane associated proteins,
namely tumor necrosis factor receptor 2 (TNFR2), MET, and ezrin. The protein of interest was
labeled either with a fluorescent protein or using a fluorescently labeled ligand of the respective
receptor. The fluorescence signal of the sample was detected using a sCMOS camera, which is
fast enough to observe dynamics in the millisecond time range. Autocorrelation curves of single
pixels were calculated and evaluated using the ImFCS plugin in ImageJ, providing two-dimensional
information on the diffusion and distribution of the studied protein [4]. Diffusion coefficients had
the same order of magnitude as those previously reported. Further studies using Imaging FCS will
enable new insights into the diffusion behavior of membrane receptors, the effect of different
conditions on this behavior, and the potential interaction of these membrane proteins with other
biomolecules.
[1] Simons K., Toomre D., (2000) Lipid rafts and Signal transduction. Molecular Cell Biology 1, 31-39.
[2] Singh A. P., Wohland T., (2014) Applications of Imaging fluorescence correlation spectroscopy. Chemical Biology 20, 29-35.
[3] Bag N., Wohland T., (2014) Imaging Fluorescence Fluctuation Spectroscopy: New Tools for Quantitative Bioimaging. Physical Chemistry 65,
225-248.
[4] Sankaran J., Shi X., Ho L.Y., Stelzer E.H.K., Wohland T. (2010) ImFCS: A software for Imaging FCS data analysis and visualization. Optical
Express 18: 25468-25481.

34
Quantum yield determination of various fluorophores in
heavy water and their application in single-molecule
localization microscopy
1Kathrin Klehs, 1Franziska Stegemann, 2Alexandre Fürstenberg, 1Christoph
Spahn, 3Ulrike Endesfelder, 1Mike Heilemann
1Institute for Physical and Theoretical Chemistry, Goethe-University Frankfurt, Max-von-Laue-
Str. 7, 60438 Frankfurt, Germany
2Department of Human Protein Sciences, University of Geneva, CMU, Rue Michel-Servet 1, 1211
Genève 4, Switzerland
3Department of Systems and Synthetic Microbiology, MPI Marburg, Karl-von-Frisch-Str. 16,
35043 Marburg, Germany
Photoswitchable fluorophores are the key tools in localization-based super-resolution
microscopy. [1] These techniques have been used extensively for unraveling biological structures
on the nanometer scale. [2] The localization precision, which is tightly connected to the spatial
resolution, thereby strongly depends on the number of photons emitted per fluorophore and
frame. [3]
Replacing water (H2O) by heavy water (D2O) was found to enhance the quantum yield of oxazine
[4] and cyanine dyes fluorescing at wavelengths above 650 nm [5]. Therefore, we also studied the
brightness of a selection of dyes under photoswitching conditions [6] on biological samples. On
average, we found an increased number of photons per single-molecule blinking event, while
using improved buffer conditions based on heavy water.
Recently, we extended our investigations towards other classes of fluorophores spread over the
whole spectral range. We found a pronounced increase in quantum yield for all fluorophores
emitting in the red region of the visible spectrum, which is of special interest, since these dyes
generally exhibit lower quantum yields, but are favored in single-molecule applications due to
reduced background from autofluorescence.
[1] M. Heilemann, P. Dedecker, J. Hofkens and M. Sauer, Laser and Photonics Reviews 2009, 3, 180 – 202.
[2] D. Kamiyama and B. Huang, Developmental Cell 2012, 23, 1103 – 1110.
[3] R. E. Thompson, D. R. Larson and W. W. Webb, Biophysical Journal 2002, 82, 2775 – 2783.
[4] S. F. Lee, Qu. Vérolet, A. Fürstenberg, Angew. Chem. Int. Ed. 2013, 52, 8948 – 8951.
[5] K. Klehs, C. Spahn, U. Endesfelder, S. Lee, A. Fürstenberg and M. Heilemann, ChemPhysChem 2014, 15(4), 637 – 641.
[6] S. van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann and M. Sauer, Nature Protocols 2011, 6(7), 991 – 1009.

35
Drebrin-like protein DBN-1 is a sarcomere component that stabilizes actin
filaments during muscle contraction
1Eugenia Butkevich, 1Kai Bodensiek, 1Nikta Fakhri, 1Kerstin von Roden, 2Iwan A.T. Schaap, 3Irina
Majoul, 1Christoph F. Schmidt, 1Dieter R. Klopfenstein
1III. Institute of Physics – Biophysics, Georg-August-University Göttingen Friedrich-Hund-Platz 1,
2Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), 37073
Göttingen, Germany.
3Institute of Biology, Center for Structural and Cell Biology in Medicine, University of Lübeck,
23538 Lübeck, Germany.
Actin filament organization and stability in the sarcomeres of muscle cells are critical for force
generation. Here we identify and functionally characterize a Caenorhabditis elegans drebrin-like
protein DBN-1 as a novel constituent of the muscle contraction machinery. In vitro, DBN-1 exhibits
actin filament binding and bundling activity. In vivo, DBN-1 is expressed in body wall muscles of
C. elegans. During muscle contraction cycle, DBN-1 alternates location between myosin- and
actin-rich regions of the sarcomere. In contracted muscle, DBN-1 is accumulated at I-bands where
it likely regulates proper spacing of a-actinin and tropomyosin and protects actin filaments from
the interaction with ADF/cofilin. DBN-1 loss of function results in the partial depolymerization of
F-actin on muscle contraction. Taken together, our data show that DBN-1 organizes the muscle
contractile apparatus maintaining the spatial relationship between actin-binding proteins such as
a-actinin, tropomyosin and ADF/cofilin and possibly strengthening actin filaments by bundling.

36
Copy number and cluster analysis of toll-like receptor 4 upon treatment with
lipopolysaccharide using single-molecule localization microscopy
1Carmen Krüger, 2Marie Theres Zeuner, 2Darius Widera, 1Mike Heilemann
1Institute of Physical and Theoretical Chemistry, Johann Wolfgang Goethe-University, Frankfurt
(Main), Germany
2School of Pharmacy, University of Reading, Reading, United Kingdom
The human toll-like receptors (TLRs) are known to play an important role in innate immunity. The
interaction of TLR4 and its ligands of the family of lipopolysaccharides (LPS) [1] is the most studied
system in this field. TLR4 is a transmembrane receptor initially located on the plasma membrane
of mammalian cells, and upon activation triggers the signaling and the initiation of immune
response through different signaling pathways. The level of response is dependent on the
respective LPS. LPS itself is a component of the outer membrane of gram-negative bacteria, e.g.
Escherichia coli. The structure of LPS varies depending on the bacterial species and leads to a
different immune response [2]; LPS derived from E.coli is known to induce a strong inflammatory
response whereas the inflammation caused by Salmonella LPS is generally weaker. Recently, it
was discovered that TLR4 is also present on adult neural stem cells and plays a role in their
neurogenesis [3].
Single-molecule fluorescence techniques are a useful toolbox to investigate assembly, activation,
organization and interaction of membrane receptors and their ligands at the single-cell level [4,
5]. Here, we investigate the activation of TLR4 in neural cells by LPS derived from E.coli and
Salmonella using single-molecule imaging. The aim of this study is to characterize the behavior of
the receptor on neural cells in the presence of different LPS species over time, which is still
unknown. Here, we quantify the number of receptor sites with respect to ligand treatment over
different time points. Furthermore, we study the size of receptor clusters using coordinate-based
and image-based analysis. Analysis of the Ripley’s H- function shows a clear maximum for the
characteristic cluster size at 60 nm for all applied conditions. For the number of particles on the
cell, we count 7 TLR4 particles per μm² in the uninduced case. Interestingly, this number only
varies slightly after treating the cells with different LPS species.
[1] Takeda, K. & Akira, S., 2005. Toll-like receptors in innate immunity. International Immunology
[2] Miller, S.I., Ernst, R.K. & Bader, M.W., 2005. LPS, TLR4 and infectious disease diversity. Nature reviews. Microbiology
[3] Okun, E., Griffioen, K.J. & Mattson, M.P., 2011. Toll-like receptor signaling in neural plasticity and disease. Trends in neurosciences
[4] Dietz, M. S., Fricke, F., Krüger, C. L., Niemann, H. H. & Heilemann, M. (2014), Receptor–Ligand Interactions: Binding Affinities Studied by
Single-Molecule and Super-Resolution Microscopy on Intact Cells. ChemPhysChem, 15: 671–676

37
[5] Fricke,F., Malkusch,S., Wangorsch,G., Greiner, J. F,. Kaltschmidt,B., Kaltschmidt,C., Widera,D., Dandekar,T.& Heilemann,M., 2014.
Quantitative single-molecule localization microscopy combined with rule-based modeling reveals ligand-induced TNF-R1 reorganization toward
higher-order oligomers. Histochemistry and Cell Biology
Mechano-sensitivity is cell type specific
Galina Kudryasheva, Florian Rehfeldt
Nowadays it is widely acknowledged that cellular function, morphology and fate are dependent
on the mechanical properties of their micro-environment. Human mesenchymal stem cells
(hMSCs) are a striking example that stem cell differentiation into various cell types can be guided
by bio-chemical cues such as growth factors or hormones as well as also by tuning the
extracellular matrix stiffness. While the entire differentiation process can take several days up to
weeks, the structure and dynamics of stress fibers can be used as an early morphological marker
and theoretically modelled using classical mechanics with an active spring model. We use this
approach to analyze the mechanical cell-matrix interactions of hMSCs and differentiated cells
(C2C12 myoblasts , SAOS-2 osteoblasts and 3T3 fibroblasts).
We plate hMSCs and differentiated cells on elastic poly-acrylamide (PA) hydrogels covering the
whole physiological range of stiffness given by Young’s moduli E from 1 to 100 kPa. Applying
immunofluorescence approach we label stress fibers and analyze cytoskeletal morphology by
fluorescence microscopy. We analyze cell shape and the alignment of stress fibers by an order
parameter as early morphological marker and extract corresponding material constants that
show distinct differences during the differentiation process. Our experiments showed that
cellular susceptibility to the substrate elasticity is highly cell type specific.

38
Super-resolution microscopy on SLAC1, its homologs and interaction partners
1Julian Lehmann, 2Dietmar Geiger, 2Rainer Hedrich, 1Markus Sauer
1Department of Biotechnology & Biophysics, Biozentrum, Julius-Maximilians-University
Würzburg, Am Hubland, 97074 Würzburg, Germany
2Department of Plantphysiology and Biophysics, Botany I, Julius-Maximilians-University
Würzburg, Julius-von-Sachs-Platz 2, 97082 Würzburg, Germany
Stomata are the doors to the outerworld of every leaf in a plant. They cater the CO2 uptake in the
leaf to ensure photosynthesis. Also, they regulate the loss of water, which is indispensable for a
plant. Stomatal movement, which is triggered by stressful stimuli like drought (e.g.) is driven by a
variation of the turgor pressure in the guard cells. Slow Anion Channel 1 (SLAC1) and its homologs
(SLAH1-SLAH4) are located in the cellular membrane of the guard cells and are responsible for
the nitrate (NO3-) an chloride (CL-) efflux, to generate osmotic pressure, which leads to an water
efflux and the closure of the stomata. SLAC1 and its homologs are activated by calcium-
dependent protein kinase (CPKs) and open stomata 1- kinase (OST1) [1].
DNA constructs of all these channels and kinases could be generated with photo-switchable/ -
convertible/ -activatable fluorescent proteins (FP), as well as with SNAP-, HALO- and CLIP-TAG.
Channels and kinases were linked with a FP/TAG at the C- and N-terminus and could be expressed
in xenopus laevis oocytes and HEK-cells. Direct stochastic optical reconstruction microscopy
(dSTORM) [2] and photoactivated localization microscopy (PALM) [3] experiments revealed high
and reproducible expression of plant anion channels (mainly in vesicles) of HEK293 cells. To
investigate the spatial dynamics of SLAC/SLAHs and their regulatory ABA signalling components
we will analyze changes in their distribution by multicolor PALM and dSTORM techniques.
[1]Geiger, D., et al., Stomatal Closure by Fast Abscisic Acid Signaling Is Mediated by the Guard Cell Anion Channel SLAH3 and the Receptor CAR1.
Science Signaling, 2011. 4(173).
[2]van de Linde, S., et al., Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nature Protocols, 2011. 6(7): p.
91-1009.
[3]Betzig, E., et al., Imaging intracellular fluorescent proteins at nanometer resolution. Science, 2006. 313(5793): p. 1642-1645.

39
Counting Receptor Channels in Membrane Patches Using Direct Stochastic
Optical Reconstruction Microscopy
Yunzhi Lu, Martin Pauli, Dmitrij Ljaschenko, Sven Proppert, Mila Paul, Anna-Leena Sirén,
Manfred Heckmann
Department of Neurophysiology, Institute of Physiology, Julius-Maximilian-University Würzburg,
Germany
Super resolution optical microscopy has improved our understanding of biology at the single
molecule level. In this study we describe a method based on direct Stochastic Optical
Reconstruction Microscopy (dSTORM) to count the number of specific receptor molecules in an
excised patch of a cell membrane. Traditionally, the number of receptor molecules is estimated
from the current measured using electrophysiological methods. However, it reports only the
number of active channels, through which ions flow upon agonist binding. It remains unclear
whether in addition to the active channels, there is a significant population of inactive channels,
which are able to bind agonists but do not respond to them. To correlate the level of active
channels with the total level of channels expressed, we performed dSTORM and
electrophysiological measurements on the same patch, which were less than 3.5 µm2 in area.
Muscle type nicotinic acetylcholine receptors (nAChRs) are among the most studied ion channels.
We transiently expressed nAChRs in HEK293 cells and first used outside-‐out patch technique.
Since nAChRs desensitize rapidly in the presence of acetylcholine (ACh), we used a rapid agonist
application system to activate these channels. After estimating the number of active channels
electrophysiologically, we labeled the receptors using Alexa-‐488 conjugated Bungarotoxin. We
mounted the patch pipettes in a custom designed rig on a dSTORM microscope and imaged the
membrane patches. The number of receptor molecules was determined by counting the
fluorescence signal localizations. We propose to explore the relationship between
electrophysiologically active channels and channels determined by dSTORM, to understand how
active receptors are related to all expressed receptors.

40
Investigating humoral autoimmunity against the NMDA-receptor NR1 subunit by
super-resolution fluorescence microscopy
Franziska Neubert, Sören Doose, Markus Sauer
Department of Biotechnology and Biophysics, Julius Maximilians University Würzburg, Germany
Anti-N-Methyl-D-aspartate receptor (NMDAR) encephalitis is a recently discovered synaptic
autoimmune disorder in which patients develop a multistage disease course with behavioral and
personality changes including psychotic episodes and deficits in memory and cognition. These
symptoms are followed by motor dysfunction and seizures that progress to loss of consciousness
to severe coma, central hypoventilation and death. The disorder predominantly affects female
children and young adults and occurs with or without tumor association (usually ovarian
teratoma). Anti-NMDA receptor encephalitis is associated with auto-antibodies in serum and
cerebrospinal fluid (CSF) against NMDARs, leading to their reversible removal from the synapse
surface. The NMDARs are heterodimers of two NR1 subunits and two NR2 (NR2A or NR2B)
subunits and play an important role in synaptic plasticity and activation of secondary intracellular
signal cascades. The pathogenic patient auto-antibodies (IgG) are mostly directed to the NR1
subunit and thereby decrease the surface density of NMDARs. The aim of the PhD project is to
investigate the influence of purified pathogenic human IgG to the NR1 subunit on the
morphological integrity and function of the NMDAR ion channel. Therefore, single-molecule
localization based super-resolution fluorescence imaging (dSTORM) is applied to evaluate anti-
NR1 auto-antibody-induced changes in biophysical properties for NMDAR ion channel function.

41
The origin of anomalous increase of the reduced viscosity in the
polyelectrolyte dilute solution
1Kengo Nishi, 2Saki Tochioka, 2 Taihei Yamada, 3Mitsuhiro Shibayama, 3Kazuki
Sada
2Institute for Solid State Physics, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba
77-8581, Japan;
3Department of Chemistry, Graduate School of Science, Hokkaido University, Kita 10, Nishi 8,
Kita, Sapporo 060-0810, Japan
In the case of neutral polymer solutions, the reduced viscosity ηSP/C is propotinal to the
concentration C in the dilute solution. This well-known relation is used for determining the
intrinsic viscosity [η] simply by extrapolating to C = 0. On the other hand, the reduced viscosity of
the salt-free polyelectrolyte solution exhibits an anomalous behavior; it increases with decreasing
C and in a range of very dilute concentrations.
In most textbooks and literature, the increase of the reduced viscosity of polyelectrolyte solutions
with dilution has been ascribed to the expansion effect of polyelectrolyte chains. On the other
hand, another explanation for this characteristic behavior was suggested in terms of
intermolecular electrostatic interaction between polyions. However, it is unclear which effect is
dominant for the viscosity of the polyelectrolyte solutions at the present stage.
In order to solve this problem, we introduced ionic groups into poly(octadecyl) acrylate1 Because
this polymer has a very long side chain, it takes stretched conformation. The point of this polymer
is that this stiff polyelectrolyte is not supposed to change its stretched conformation due to the
electrostatic repulsion. We evaluated the chain conformation of poly (octadecyl) acrylate with
ionic groups from small-angle neutron scattering2. By comparing with the result of the viscosity
Measurement3 , we determined which effect is important for the viscosity of the polyelectrolyte
solutions, i.e., the expansion effect or the intermolecular electrostatic interaction.
1. T. Ono, T. Sugimoto, S. Shinkai and K. Sada, Nat. Mater. 6, 429-433 (2007).
2. K. Nishi, S. Tochioka, T. Hiroi, T. Yamada, T. Kokado, T. H. Kim, E. P. Gilbert, K. Sada and M. Shibayama, Macromolecules 48, 3613-3621 (2015).
3. T. Ono, M. Ohta and K. Sada, ACS Macro Lett. 1, 1270-1273 (2012).

42
Mechanotransduction in the pentamere organ of the Drosophila larva
Achintya Prahlad
The fruit fly Drosophila melanogaster uses mechanosensation for several purposes. One class of
specialized organs are the chordotonal organs, such as the antennal auditory organ of the adult,
and the larval pentamere organ (or lch5). The sensory neurons at the core of these organs have
one dendrite, which terminates in a cilium. The cilia are believed to be the main
mechanotransducers. The lch5 organ aids in locomotion by giving feedback to the central nervous
system. We focus on this organ because its sensory neurons are well accessible to manipulation
under the microscope.
Some molecular and anatomical aspects of these organs have been studied. However, an
understanding of the internal transduction mechanics and the manner in which membrane
channels are activated upon deflection of the cilium is still elusive. We are using a preparation of
the larva under buffer solution that allows us to directly contact the sensory neurons of the lch5.
Our approach is to provide controlled mechanical stimuli to the organ and measure the
mechanical and electrical response.

43
Small Labeling Pair for Single-Molecule Super-Resolution Imaging
1Anika Raulf, 2Ralph Wieneke, 2Alina Kollmannsperger, 2Robert Tampé, 1Mike Heilemann
1Institute of Physical and Theoretical Chemistry, Goethe-University Frankfurt, Germany
2Institute of Biochemistry, Goethe-University Frankfurt, Germany
In fluorescence microscopy a target protein is not directly detected, rather it is fluorescently
labeled and this fluorophore is localized. Thus several labeling strategies are available; widely-
used are immunostaining, fluorescent fusionproteins or protein tags which can be stained
afterwards (e.g. SNAP- or Halo-tag). However, all these techniques have one or more drawbacks
(low quantum yield, sterical constrains of fusion domains, large distance between label and target
protein, insufficient specificity or reactivity).
Here, we used a small labeling pair (SLaP) consisting of the genetically encoded His-tag and a Ni-
trisNTA coupled fluorophore to label our target proteins1. Ni-trisNTA has a high affinity for His-
tagged proteins (KD ≤ 10 nM)2; to further confirm a high target specificity, we used cytoskeleton
proteins fused with fluorescent proteins and a His-tag which was labelled via SLaP. Confocal laser
scanning microscopy shows a nearly perfect colocalization of the fluorescent protein and the
organic dye attached via SlaP.
Direct stochastic optical reconstruction microscopy (dSTORM)3 of cytoskeleton proteins labeled
via SLaP, reveals actin filaments with widths down to 40 nm as well as a high spatial resolution
for laminA at the nuclear envelope. These results clearly demonstrate the suitability of SLaP for
single-molecule localization techniques. Further, we performed dSTORM imaging of an ABC
transporter located in the ER membrane (transporter associated with antigen processing; TAP). A
comparison between immunostained and SLaP labeled TAP shows a difference of 22 nm in cluster
size, which is in agreement with the localization inaccuracy caused by two antibodies (2 x 10 nm),
since the size of the small labeling pair is approximately 1 nm.
In conclusion, labeling via SLaP is highly specific, allows the use of bright organic dyes suitable for
single-molecule super-resolution microscopy and minimizes localization inaccuracy due to its
small size.
[1]Wienecke, Raulf, Kollmannsperger, Heilemann and Tampé, Angew. Chemie. Int. Ed. (2015).
[2]Lata, Reichel, Brock, Tampé and Piehler, J. Am. Chem. Soc. (2005).
[3]Heilemann, van de Linde, Schüttpelz, Kasper, Seefeldt, Mukherjee, Tinnefeld and Sauer, Angew. Chemie Int. Ed. (2008).

44
Viscoelastic mechanics of non-adhering cells
Samaneh Rezvani, Christoph F. Schmidt
Cells sense their micro-environment through biochemical and mechanical interactions. They can
respond to biochemical and mechanical stimuli by undergoing shape- and possibly volume
changes. The key components in determining the mechanical response of a cell are the
viscoelastic properties of the actomyosin cortex, effective surface tension, and the osmotic
pressure. We probe suspended rounded-up cells by active and passive microrheology to describe
the roles of the various components.

45
Improvements for Stochastic Optical Fluctuation Imaging (SOFI):
Sub-pixel super-resolution images with a conventional wide-field microscope
Simon Christoph Stein, Anja Huss, Jörg Enderlein
The last decade has seen a rapid evolution of a wide array of new super-resolution microscopy
techniques which are by now widely available and applied in the life sciences. Among these
different techniques, super-resolution optical fluctuation imaging (SOFI) [1] stands out due to its
algorithmic and experimental simplicity, requiring only the rapid recording, with a conventional
wide-field setup, of the intensity fluctuations from a sample which is labeled with blinking
emitters.
The visual fidelity of SOFI, however, is limited by the finite size of the camera’s pixel grid: Recent
papers have proposed to use the spatio-temporal cross-correlations between different pixels to
add virtual sub-pixels to the grid [2]. Major drawbacks of this approach are the increasing
algorithmic complexity with increasing correlation order and the lack of a canonical way to do the
calculations. This easily introduces artifacts to the final image. Here, we present a new approach
for creating high-resolution images which is based on up- sampling the image stack in Fourier
space before the SOFI algorithm is applied. The calculation of the nth order SOFI image leads to
an n-times enlargement of available spatial frequencies in Fourier space, which are captured by
the finer pixel grid. The method is completely artifact-free and straightforward to implement,
keeping the simplicity of the original algorithm.
[1] Dertinger, T.; Colyer, R.; Iyer, G.; Weiss, S. & Enderlein, J. Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI),
Proceedings of the National Academy of Sciences, 2009, 106, 22287-22292
[2] Dertinger, T.; Colyer, R.; Vogel, R.; Enderlein, J. & Weiss, S. Achieving increased resolution and more pixels with Superresolution Optical
Fluctuation Imaging (SOFI). Optics express, 2010, 18, 18875-18885

46
Mechanical coupling between the cytoskeleton and the nucleus
Gabriele Straass, Florian Rehfeldt
It is nowadays widely acknowledged that mechanical cues are as important for cellular behavior
as traditional biochemical ones. Strikingly, adult stem cells can be guided to differentiate
towards various cell types when cultured on elastic hydrogels with appropriate Young's
modulus E. While the differentiation process takes several days, the acto-myosin cytoskeleton
organization shows significant differences within the first 24 hours after plating. We investigate
the mechanical properties of the nucleus by atomic force microscopy and fluorescence
microscopy and demonstrate the impact of substrate elasticity E on nuclear morphology via
acto-myosin stress fibers. Elucidating the mechanical coupling of the cytoskeleton and the
nucleus might reveal a direct mechanical pathway that alters gene transcription and might
impact adult stem cell differentiation.

47
Measuring diffusion on the nanometer scale with Single-Molecule Metal
Induced Energy Transfer (smMIET)
Jan Thiart, Jörg Enderlein
When an electric dipole emitter such as a fluorescent molecule is brought close to a metallic
structure, plasmonic coupling to the metal’s electrons causes a dramatic change in its
fluorescence lifetime. Single-Molecule Metal Induced Energy Transfer (smMIET) employs this
effect for determining the axial distance of a fluorophore to a metallic surface with nanometer
precision. Here, we demonstrate how smMIET can be used to investigate lipid diffusion in small
unilamellar vesicles (SUVs) with sizes from 30 nm to 100 nm diameter, much smaller than the
typical spatial resolution of a fluorescence microscope. The SUVs are placed above a gold
nanolayer, and the intensity fluctuations generated by a fluorescently labeled lipid moving
through the vesicle are recorded. The diffusion coefficient can then be directly extracted with
a fluorescence correlation analysis. By measuring diffusion coefficient or different sizes of SUV,
we obtain a relation between lipid membrane diffusion and curvature. The obtained results will
be important for a better understanding of curvature effects on membrane diffusion in
biological systems.

48
Light-induced cell damage in live-cell super-resolution microscopy
Sina Wäldchen, Julian Lehmann, Teresa Klein, Sebastian van de Linde, Markus Sauer
Department of Biotechnology and Biophysics, Julius Maximilians University Würzburg, Germany
Super-resolution microscopy can unravel previously hidden details of cellular structures but
requires high irradiation intensities to use the limited photon budget efficiently. Such high
photon densities are likely to induce cellular damage in live-cell experiments. We applied
single-molecule localization microscopy conditions and tested the influence of irradiation
intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling
on the survival probability of different cell lines 20-24 hours after irradiation as well as the
influence on microtubule growth. The photo-sensitivity is dramatically increased at lower
irradiation wavelength. We observed fixation, plasma membrane permeabilization and
cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light
intensities of ~ 1 kW cm-2 at 640 nm for several minutes, the maximum dose at 405 nm is only
48 J cm-2, emphasizing red fluorophores for live-cell localization microscopy. We also present
strategies to minimize phototoxic factors and to maximize the cells ability to cope with higher
irradiation intensities.

49
Structure formation dynamics of stress fibers in adult stem cells
1Carina Wollnik, 2Benjamin Eltzner, 2Stephan Huckemann, 1Florian Rehfeldt
2Institute for Mathematical Stochastics, Georg-August-University, D-37077 Göttingen, Germany
Adult human mesenchymal stem cells (hMSCs) differentiate into cell types like nerve, bone or
muscle precursor cells. During differentiation, orchestration of biological and chemical cues is
needed, but substrate stiffness alone is enough to guide hMSCs towards different lineages in the
absence of additional biochemical stimuli as shown by Engler et al. [1]. Stress fibers composed of
actin _laments, cross-linkers and myosin motor-proteins, generate and transmit forces
throughout the cell [3]. Inhibition of myosin stops the differentiation process [1], implying
importance of stress fiber tension for differentiation. Characteristic reorganization of stress fibers
can be detected within 24 hours and used as an early morphological marker [2]. Interestingly,
there is a non-monotonic dependence of stress fiber polarization on the Young's modulus of the
underlying substrate [2]. Performing 24 h live-cell imaging of RFP-Lifeact transfected hMSCs on
substrates of different stiffness and tracing of stress fibers with sophisticated _lament tracking
algorithms [3,4], we gain a deeper understanding of stress fiber formation dynamics in early stem
cell differentiation.
[1] A. Engler et al., Cell (2006)
[2] A. Zemel et al., Nature Physics (2010)
[3] E.K. Paluch et al., BMC Biology (2015)
[4] B. Eltzner et al., PLoS ONE (2015).
[5] S.F. Huckemann et al., Bernoulli, arXiv.org: 1404.3300 (2014). In press.

50
Conference information

51
Bus transfer Göttingen to Frankfurt Airport
• Wednesday, 07.10.2015
• Red arrow: Meeting point
• Bus will depart at 22.45 straight
• Return: Monday, 12.10.2015, approximately 3 am
Flights:
• Frankfurt International Airport Terminal 2
• Departure flight AB7772, 08.10.2015 at 4.45 am
• Return flight AB7751, 11.10.2015 at 8.25 pm
• Luggage: http://www.airberlin.com/en-DE/site/landingpages/baggage_services.php
• Directions: http://www.airberlin.com/en-DE/flughafen-frankfurt
• Boarding pass: Issuing at the Airport (Air Berlin counter). Show your ID card / Passport and
use the ticket number 6N28CC to receive your boarding pass.

52
All-inclusive rules
1. On arrival you will be given a card with your picture and that will be your all-inclusive-card.
2. Please show your all-inclusive-card at Bars and restaurants of Resort.
3. Your card is personal and not transferable to other guests. If you want to invite someone you
need to pay full price.
4. Your all-inclusive-card will be valid from arrival until 12:00 midday on your departure day. And
you can use your all-inclusive-card from 07:30h till 24:00h.
5. Please finish your drink before ordering another.
6. Conditions: all members of the same reservation and room must have the same board basis.
What is included?
• Breakfast Buffet: from 07:30h to 10:00h in Restaurants Ancora and Denario.
• Afterhours Buffet: continental breakfast from 10:30h to 11:30h at the Grill Aquarius.
• Lunch: 12:00h to 16:00h in our a la carte Grill Aquarius or buffet from 12:30h till 14:00h at
Restaurant Denario.
• Dinner: from 18:30h to 22:00h at buffet restaurant Denario and Ancora.
• Thematic dinner: 2 times a week at buffet restaurant Denario and Ancora (Eastern, Italian,
French, Spanish, Mallorcan, etc.)
• Snacks: hot and cold snacks from 10:00h till 12:00h and 16:00h to 18:00h in Restaurant Grill
Aquarius.
• Drinks: you can enjoy a good selection of drinks all day from 10:00h to 24:00h.
• In our Resort all soft drinks and alcoholic beverages are served to the table.
• Bar Luna and Triton time table is from 10:00h to 24:00h.
• Mini-bar in rooms with soft drinks and beer are filled up every day.
• Free access to sauna, turkish bath, jacuzzi and gym.

53
Hiking:
Parc natural de S’Albufera de Mallorca: Spectacular marsh,
the everglades of Mallorca. The S'Albufera Natural Park is
possibly the most extensive and bird-rich wetland to be
found on any mediterranean island. Its 1,646.5 ha have
enjoyed protection since 1988 and now have a visitor's
reception area, a permanent exhibition and a good number
of hides, observation platforms and marked itineraries.
Formentor: Spectacular views from high cliff. Cap de Formentor is a spectacular place, located on the
northernmost point of the Balearic Island Mallorca in Spain. Its highest point, Fumart, is 384m above sea
level. It has many associated bays, including Cala
Fiquera, Cala Murta and Cala Pi de la Posada.The 13.5
km road which runs from Port de Pollença to Cap de
Formentor was built by the Italian engineer Antonio
Parietti. His masterpiece on Mallorca, however, was
the snake to Sa Calobra. Instead of being overwhelmed
by what stood in his way on the cliffs, Parretti observed
the Tramuntana winds and understood: where the
slope was too steep, he made a curve. When he had to
remove part of the cliffs, he placed the waste in other places where it was needed. The result was the two
roads, which are nestled together in the mountains like abandoned silk ribbons.

54
Biking:
The hotel has a huerzler.com bike station one can
rent road bikes and helmets. Please bring your own
cycling shoes and check if they fit to the pedal
system. They don’t rent out Mountain bikes. Go on
their webpage to reserve your bike and equipment
if you like. Mallorca is the winter training arena of
all professional cyclist, for the enthusiast it’s a must
cycling on their roads. If one fears the Spanish traffic, be advised to cycle in groups.
http://www.huerzeler.com/en/cycling-stations/detailview-radsportstationen/?hID=11
Culture and history:
Alcudia: Visit the old roman village and historic center of
Alcudia, there will be a market every Tuesday and Sunday
morning.
Pollentia: One can combine a visit at the market, held
every Sunday morning with a walk in the historic village.
Catamaran tour:
The port of Alcudia is the starting point to some of the
greatest catamaran tours on the island, please check their
webpage if you are interested in this water experience.
http://www.click-mallorca.com/ausfluge
sehenswurdigkeiten/puerto-alcudia/katamarantour-
alcudia/#.VhFzocvosyl

Pulsed Lasers
Picosecond Timing
Confocal Microscopes
Fluorescence Spectrometers It's about time.
Features
• STED resolution below 50 nm
• STED laser at 765 nm
• FLIM, FRET, FCS, FCCS, FLCS
• 2D STED-FCS/FLCS
• Gated STED (gSTED) and gSTED-FCS
MicroTime 200: An all-in-one solution for time-resolved
microscopy with unmatched sensitivity
Further extensions
• Atomic Force Microscopy
• Wide-field microscopy
• Spectrograph
• Cryostat
• 2-focus FCS
PicoQuant GmbH
Berlin, Germany
info@picoquant.com
www.picoquant.com
Super-resolution
Microscopy
STED Add-on for the MicroTime 200
FLIM and FCS Platform

FSM 2015 - International Biophysics Conference

Recommended

Recommended

More Related Content

What's hot

What's hot (19)

Viewers also liked

Viewers also liked (20)

Similar to FSM 2015 - International Biophysics Conference

Similar to FSM 2015 - International Biophysics Conference (20)

More from Dirk Hähnel

More from Dirk Hähnel (8)

Recently uploaded

Recently uploaded (20)

FSM 2015 - International Biophysics Conference