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7th ASEM-Workshop

Advanced Electron Microscopy

April 20th – 21st, 2017

Venue: Technische Universität Wien TUtheSky, Getrteidemarkt 9, 1060 Wien

We cordially thank our sponsors:

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Scope of the workshop

This workshop is a meeting for electron microscopists and all those interested in electron microscopy

and its applications in modern science and technology. Students and scientists from all fields of

microscopy in life sciences, materials science and physical science are welcome. The goal of this

workshop is to join student and expert scientists. It provides an Austrian-wide discussion forum for

the work done in academia and industrial based research. As a platform for oral presentations by

young scientists, this year it is especially oriented in view of the two big European conferences in

Lausanne and Rovinj, the MC2017 and the MCM2017, respectively.

The conference language is English.

Local Organizers

Prof. Johannes Bernardi

University Service Centre for Electron Microscopy (USTEM), Technische Universität Wien

Wiedner Hauptstraße 8-10, 1040 Vienna

(T) +43 (0) 1-58801-45210

(F) +43 (0) 1-58801-9-45210

(E) bernardi@ustem.tuwien.ac.at

Prof. Michael Stöger-Pollach

University Service Centre for Electron Microscopy (USTEM), Technische Universität Wien

Wiedner Hauptstraße 8-10, 1040 Vienna

(T) +43 (0) 1-58801-45204

(F) +43 (0) 1-58801-9-45204

(E) stoeger@ustem.tuwien.ac.at

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TUtheSky

Getreidemarkt 9, 1060 Wien

Lageplan

Mit dem Aufzug in das 11. Stockwerk fahren!

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Programme

Thursday, 20th April 2017

13:00 – 13:25 Registration

13:25 – 13:30 Opening

Session 1 – Life Science

13:30 Dietmar Pum (invited)

S-layer proteins

14:00 FEI – Firmenvortrag: Ben Lich

Cryo EM workflows for Single Particle Analysis and Tomography of hydrated, intact cells

14:15 Philipp Steiner

Stress induced fusion of mitochondria visualized by electron tomography in plants

14:30 Sabrina Oberwegser

Tsunamia transpacifica – TEM investigations in a newly-discovered red algal genus

colonizing Japanese tsunami debris

14:45 Margret Eckhard

How to preserve a moss for element analysis? Different ways of preparation for electron

microscopy

15:00 Daniel Serwas

How Cells Build Their Antenna: Centrioles Initiate Cilia Assembly, But Are Dispensable for

Cilia Maturation and Maintenance

15:15 David Kleindienst

Compartment-specific association of GABAB receptors and their effector ion channels in

cerebellar Purkinje cells

15:30 Gatan – Firmenvortrag: Andreas Kastenmüller

Recent technology improvements for Electron Microscopy

15:45 – 16:15 Coffee break

Session 2 – Materials Science

16:15 Fritz-Grasenik Preisvortrag: Markus Herbst Characterization of the Vasa vasorum in the human great saphenous vein by SEM and 3D-morphometry of vascular corrosion casts

16:45 Tia Truglas

The effects of double annealing on medium manganese steel

17:00 Tomasz Wojcik

Phase characterization in Ni-base superalloy Rene 65

17:15 Christian Ebner

Viscoelastic stress relaxation of TiAl thin film under tension measured by selected area

electron diffraction

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17:30 JEOL – Firmenvortrag: Georg Raggl

The JIB-4700F - JEOLs new high performance FIB multi-beam system

17:45 Ulrich Haselmann

HRTEM study of Ca doped Bismuth Ferrite

18:00 Bernhard Bayer

Introducing overlapping grain boundaries in chemical vapor deposited hexagonal boron

nitride monolayer films

18:15 Stefan Pfeiffer

Combined analytical TEM and magnetic investigation of the effects of neutron irradiation

on Nb3Sn superconductors

19:00 – 21:30 Workshop dinner

Friday, 21st April 2017

Session 3 – Life Science

08:30 Zaoli Zhang (invited)

Advanced characterization of materials using atomic resolution TEM

09:00 Mariella Sele

High Resolution Visualisation of Iron Deposits in the Human Brain in Health and Disease

09:15 Christoph Dibiasi

Impact of fibrinogen concentration on blood clot formation

09:30 Leica – Firmenvortrag: Robert Ranner

Optimized sample preparation by using a correct workflow

09:45 Virginie Hubert

Using electron microscopy as a method to monitor autophagy

10:00 Stefan Schulz

Serum derived exosomes as a putative diagnostic tool for ANCA associated vasculitis

10:15 Carolina Borges-Merjane

Flash and Freeze: combining high-pressure freezing and optogenetics to evaluate

synaptic transmission

10:30 Jacek Plewka

SEM on agarose-based chromatographic beads – how to recalculate a reference SAXS

scattering signal from an image

10:45 – 11:15 Coffee break

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Session 4 – Materials Science

11:15 Michal Horak

Babinet principle for plasmonic antennas: complementarity and differences

11:30 Franz-Philipp Schmidt

Hybrid plasmonics: From plasmon-plasmon to plasmon-exciton coupling

11:45 Wolfgang Wallisch

Influences of the CMR effect on dielectric properties

12:00 Semir Tulic

Reaction of Ni and C thin films studied by TEM and SEM

12:15 ZEISS – Firmenvortrag: Wolfgang Schwinger

Analytical FIB-SEM Tomography without Compromises

12:30 Robert Sriemitzer

Dealing with light refraction in 3D mapping in combined Raman/SEM

12:45 Philipp Siedlacek

SEM Characterization of functionalized Carbon Nanotubes

13:00 Robert Winkler

FEBID Based Direct-Write of 3D Plasmonic Gold Structures

13:15 Jürgen Sattelkow

Direct-Write Fabrication of Electric and Thermal High-Resolution Nanoprobes on Self-

Sensing AFM Cantilever

13:30 Snacks and Farewell

Poster:

Martin Meischel

Nanoscale studies of mechanical properties of rat bones around biodegradable implants

Stefan Löffler

Convergent-Beam EMCD: Efficient Magnetic Measurements on the Nanoscale

Thomas Schachinger

Vortex Filter EMCD: Towards an Alternative EMCD Approach

Christoffer Müller

Flash-annealed CuZr based bulk metallic glass studied by electron microscopy methods

Harald Fitzek

Understanding surface enhanced Raman spectroscopy using accurate simulations of

electric nearfields

Cornelia Trummer

Preparation of Transmission Electron Microscopy Samples by Mechanical Techniques in

Combination with Low-Voltage Ion Milling

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Daniel Knez

In situ studies of high-purity mono- and bimetallic nanostructures in experiment and

simulation

Manfred Nachtnebel

Polymer fracture – What can the 3D reconstruction of the crack region tell us about the

microscopic fracture mechanisms

Martina Dienstleder

Challenges in sample preparation for HRSTEM analysis

Angelina Orthacker

Investigation of the non-equilibrium formation of stoichiometric precipitates in multi-

component aluminium alloys

Stefan Geyer

High resolution episcopic microscopy (HREM): a tool for 3D imaging of organic materials

Thomas Götsch

The Electronic Phase Diagram of YSZ

Stefan Noisternig

Lamellae in FeAl deformed under hydrostatic pressure

Katharina Keuenhof

Preparation Methods of Biological Samples: a Comparison of Chemical Fixation and

High-Pressure Freezing (HPF)

Eveline Fisselthaler

Quantitative Analysis of Internal Interfaces: Structural and quantitative analysis via High

resolution STEM

Walid Hetaba

In-situ electron microscopy for heterogeneous catalysis

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S-layer proteins

Dietmar Pum(1), Uwe B. Sleytr (1)

(1) University of Natural Resources and Life Science, Vienna, Department of Nanobiotechnology, Institute of Biophysics, Muthgasse 11, 1190 Vienna, Austria

Crystalline bacterial cell surface layer (S-layer) proteins are one of the most abundant biopolymers on

earth and form the outermost cell envelope component in a broad range of archaea and bacteria

(Fig.1) [1, 2]. These S-layer protein lattices represent the simplest biological membranes developed

during evolution. S-layer lattices are highly porous protein mesh works with unit cell sizes in the range

of 3 to 30 nm and thicknesses of ∼10 nm. One of the key features of S-layer proteins is their intrinsic

capability to form self-assembled mono- or double layers in suspension, at solid supports, the air-water

interface, planar lipid films, liposomes, nanocapsules, and nanoparticles.

Basic research on S-layer proteins enabled us to use the unique self-assembly properties of native and,

in particular, genetically functionalized S-layer fusion protein lattices as matrices for the binding of

molecules and the synthesis of nano materials. In addition, most recently S-layer proteins were used

as scaffolds for making hybrid organic-inorganic nanostructures.

This contribution summarizes the state-of-the art in the reassembly of S-layer proteins, their non-

classical pathway of matrix assembly, and application as templates in the controlled deposition of

inorganic materials, such as biogenic silica.

Figure 1. (a) TEM micrograph of a freeze-etched and metal shadowed preparation of a bacterial cell of Lysinibacillus sphaericus with an S-layer (SbpA) as the outermost cell envelope component. The S-layer exhibits square (p4) lattice symmetry. The numerous lattice faults are a consequence of the bending of the S-layer lattice at the rounded cell poles. In addition, the rope-like structures are the flagella of the bacterial cell. Bar, 200 nm. (b) Atomic force microscopical image of a monolayer of SbpA S-layer proteins reassembled on a silicon surface. Image data were complemented with TEM tomography data. Unit cell size is 13.1 x 13.1 nm.

[1] Sleytr, U.B., Schuster, B., Egelseer, E.M., & Pum, D. (2014) FEMS Microbiol Rev, 38, 823-864.

[2] Pum, D., Sleytr, U.B. (2014) Nanotechnology, 25, 312001.

We kindly acknowledge financial support by the Air Force Office of Scientific Research (AFOSR)

[FA9550-15-1-0459].

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Cryo EM workflows for Single Particle Analysis and Tomography of hydrated,

intact cells

W. Voorhout, M. Storms, G. van Duinen, J. Lengyel, M. Vos and B. Lich

FEI Company, Achtseweg Noord 5, 5651 GG Eindhoven, The Netherlands

A new frontier exists in unraveling interactive biological and biochemical processes and pathways at

the macromolecular level. Of critical importance is the three-dimensional visualization of

macromolecular structures and molecular machines in their native functional state. Three techniques

play a major role, NMR, XRD and Cryo-TEM.

Nuclear magnetic resonance (NMR) has the capability to study specific protein domains or fragments

and their functional role in protein folding and dynamics and in ligand binding whereas X-Ray

crystallography (XRD) allows visualizing high-resolution but more static 3D structures of apo and

liganded proteins, mainly in a monomeric or dimeric state after crystallization. To unravel more

physiologically relevant situations however, it is essential to visualize multimeric complexes in their

tertiary and quaternary state and their interaction with other complexes. Cryo-TEM applications like

single particle analysis one can visualize multimeric complexes. In this so-called translational

methodology, cryo-TEM thus provides complementary information to NMR and XRD that can be crucial

for a detailed structural analysis for a better understanding of the mechanism of the physiologically

relevant complex.

Latest developments in the cryo-TEM workflow have brought the 3 major structural biology

technologies closer together. Now, finally, a continuum has been reached on all important aspects

with regards to resolution and macromolecular scales which allows for the full deployment of the

combination of these technologies.

We will discuss the future of structural biology based on the latest developments of the FEI workflow

and its components.

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Stress induced fusion of mitochondria visualized by electron tomography in

plants

P. Steiner (1), M. Luckner (2), G. Wanner (2) and U. Lütz-Meindl (1)

(1) University of Salzburg, Cell Biology Department, Hellbrunnerstraße 34, A-5020 Salzburg, Austria (2) Ludwig-Maximilians-University Munich, Faculty of Biology, Ultrastructural Research, Großhadernerstr. 2-4,

D-82152 Planegg-Martinsried, Germany

Physiological and molecular reactions of plant and animal cells to stress are well known from numerous

investigations. However, there is a tremendous lack in information on sub-structural alterations of

organelles that accompany stress induced processes. In higher plants and algae, structural stress

hallmarks have been reported in organelles like dictyosomes and chloroplasts [1, 2]. Advanced electron

microscopic techniques, such as electron tomography, have already provided evidence for structural

changes of mitochondria as consequence of stress or disease, both in plant and animal cells. [3, 4]

Alzheimer disease for example causes sub-structural alterations of the inner and outer membrane of

mitochondria in transgene mouse brain [4]. In the present study we investigate structural effects of

mitochondria in the unicellular freshwater alga Micrasterias denticulata after exposure to KCl by

means of FIB/SEM and TEM tomography. Whereas mitochondria in untreated control Micrasterias

cells are single, spherical or slightly elongated organelles, 3-D reconstructions of KCl exposed cells show

that they form 3-dimensional aggregates during stress. The membranous connections by which the

aggregates are formed are established by elongation of the outer mitochondrial membrane. In this

way mitochondria do not only fuse with each other but also with degenerated dictyosomes. As the

mitochondrial respiration potential of KCl stressed cells was almost the same as in controls [5] and as

the sub-structural alterations were reversible, we assume that mitochondrial aggregation is important

for maintaining essential cellular functions such as respiration during stress. We obtained similar

effects on mitochondria in the aquatic higher plant Lemna sp. after KCl exposure.

Figure 1: Protuberance of the outer

mitochondrial membrane induced by

150 mM KCl in Micrasterias denticulata.

(a) TEM micrograph, (b) reconstruction

from TEM tomography series. Protrusion

in orange.

[1] Lütz-Meindl, U., Luckner, M., Andosch, A. & Wanner, G. (2015) Journal of Microscopy, 263, 129-141. [2] Santos, C. L., Campos, A., Azevedo, H. & Caldeira, G. (2001) Journal of Experimental Botany, 52, 351–360. [3] Vartapetian, B. B., Andreeva, I. N., Generozova, I. P., Polyakova, L. I., Dolgikh, Y. I., Stepanova, A. Y. (2003) Annals of Botany, 91, 155-172. [4] Choi, K. J., Kim, M. J., Je, A. R., Jun, S., Lee, C., Lee, E., Jo, M., Huh, Y. H. & Kweon, H. (2014) Journal of Biosciences, 39, 97–105. [5] Affenzeller, M. J., Darehshouri, A., Andosch, A., Lütz, C. & Lütz-Meindl, U. (2009) Journal of Experimental Botany, 5, 854-855.

1 µm

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Tsunamia transpacifica – TEM investigations in a newly-discovered red algal

genus colonizing Japanese tsunami debris

Sabrina Obwegeser (1), John West (2), Ursula Lütz-Meindl (3), Andreas Holzinger (1)

(1) University of Innsbruck, Department of Botany, Functional Plant Biology, 6020 Innsbruck, Austria

(2) School of Biosciences 2, University of Melbourne, Parkville, VIC 3010, Australia

(3) University of Salzburg, Cell Biology Department, 5020 Salzburg, Austria

On March 11, 2011 a tremendous earthquake off the pacific coast of Tohoku shook the northeastern

shore of Japan, unleashing 40m high tsunami waves leading to the worldwide known nuclear

meltdown in the Fukushima nuclear power plant. Large amounts of debris covered the land surface

and 5 million tons were washed into the ocean [1]. Debris is still (2017) carried by the North Pacific

Current and travels a distance of about 7200 km to the west coast of North America and can be found

in Oregon and Washington since 2013. In 2015 small plastic debris carrying dense pink crusts of algae

were recovered, where a sample was then isolated. The culture was analyzed by three-gene phylogeny

and revealed a new genus and species of the red algal class Stylonematophyceae, Tsunamia

transpacifica, referring to its origin [1]. The cells have a massive wall and thick extracellular matrix of

complex polysaccharides, a single central nucleus and a purple to pink multi-lobed parietal plastid

lacking a pyrenoid [1]. The first attempts to prepare the new genus for transmission-electron

microscopy by standard chemical fixation protocols were not successful. Preservation of the cells did

not allow distinguishing cellular components, except for the massive cell walls and extracellular matrix,

which may potentially lead to a deficient infiltration of the specimens during fixation and/or

embedding processes. This is surprising, as in other marine and freshwater red algae standard

protocols were successful [2, 3]. Therefore, we used a high pressure freezing and freeze substitution

protocol, using 2% OsO4 and 0.05% uranyl acetate for postfixation during the substitution as earlier

described [4]. This yields a high quality fixation of the ultrastructure of T. transpacifica and cellular

structures not described before in the class of Stylonematophyceae. Various vacuoles as well as

unknown electron dense bodies surrounding the nucleus were found. While such electron dense

bodies are frequently observed in brown algae, where they are described as physodes, e.g. in the arctic

Saccharina latissima [5], they are uncommon for red algae. Physodes are described as phorotannin-

containing bodies and due to their spectral properties have well characterized protective functions

against UV radiation. Insights in the ultrastructure of T. transpacifica contribute to a detailed

morphological knowledge and might contribute to an understanding of their adaptations to extreme

living conditions when colonizing floating plastic debris. Electron energy loss spectra (EELS) provide

insights into the chemical composition of the electron dense bodies in T. transpacifica and indicate

elevated levels of phosphorus and cobalt.

[1] J. A. West, G. I. Hansen, T. Hanyuda, G. C. Zuccarelllo (2016) Algae, 31, 1-13 [2] A. Holzinger, U. Karsten, C. Lütz, C. Wiencke (2004) Plant Biol. 6, 568-577

[3] S. Aigner, A. Holzinger, U. Karsten, I. Kranner (2017) Eur. J. Phycol., DOI: 10.1080/

09670262.2016.1274430 [4] N. Aichinger, U. Lütz-Meindl (2005), J Microsc. 219, 86-94 [5] A. Holzinger, L. Di Piazza, C. Lütz, M. Y. Roleda (2011) Phycol. Res., 59, 221-235

We kindly acknowledge financial support by the Austrian Science Fund (FWF):[I 1951-B16]

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How to preserve a moss for element analysis? Different ways of preparation

for electron microscopy

Margret Eckhard(1), Siegfried Reipert(1), Ingeborg Lang(1) (1) University of Vienna, Core Facility Cell Imaging and Ultrastructure Research (CIUS), Althanstraße 14 1090

Vienna

margret.eckhard@unet.univie.ac.at

In my master study, I look for the best preservation method for electron energy loss spectroscopy (EELS) in the moss Physcomitrella patens. The final aim is the quantification and localization of zinc that is stored in the plant cells. I am using the moss Physcomitrella patens because it is a well-known model organism; it is easy to cultivate in the laboratory and it is very tolerant to heavy metals [1], thus it is the perfect study object for my research. However, the moss cell wall is a tight barrier and therefore challenging for electron microscopic preparations.

To achieve this goal, I am combining various methods from light and electron microscopy. At the

electron microscopy level, I compare preparation protocols for chemical fixation and cryofixation using

the Leica HPM 100 and the Leica AFS 2 with a new agitation module for accelerated freeze substitution

[2].

By now, really good fixation results were established by using high-pressure freezing combined with

rapid freeze substitution. The probes are presently used for the element analysis and zinc detection in

the transmission electron microscope. To adapt the chemical fixation protocol, previous light

microscopic observations showed very divers reactions to the provided fixation buffers by the

gametophore and the protonemata of Physcomitrella patens, so that it is necessary to adjust the buffer

depending on the part of the moss that should be preserved.

[1]Sassmann, S., et al. (2015) Environmental and experimental botany, 118, 12-20

[2] Goldammer, H., et al. (2016) Protist, 167/4, 369- 376

C

CW

Figure 1 Physcomitrella patens. A: overview of gametophyte; B: gametophore leaf, cryofixed and freeze substituted. Gametophore (G), protonemata (P), vacuole (V), chloroplast (CL), starch (S), cytoplasm (C), cell wall (CW).

A B

P

G

S

V

CL

CW

C

2µm 200µm

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How Cells Build Their Antenna: Centrioles Initiate Cilia Assembly, But Are Dispensable for Cilia Maturation and Maintenance

Daniel Serwas(1), Alexander Dammermann (1)

(1) Max F. Perutz Laboratories, University of Vienna, Vienna, Austria

In order to fulfill their versatile functions, cells need to be able to receive and transmit external

signals. Most human cells possess an evolutionarily conserved antenna-like structure on their

surface, the primary cilium, which participates in these processes.

Cilia form from centriole-derived basal bodies that serve as a platform for the assembly of multiple

structures including transition fibers, transition zone and axoneme. While it is clear that cilia

assembly is absolutely dependent on the presence of centrioles, it is not known whether centrioles

only trigger ciliogenesis or actively participate in downstream events. We used the nematode worm

C. elegans as an experimental model to address this question, since centriolar structures do not

persist at the base of mature cilia, but rather degenerate during ciliogenesis. Ciliary structures which

form after centriole loss clearly cannot be directly dependent on centrioles.

Using a combination of light microscopy and electron tomography, we generated the first timeline of

ciliogenesis. We found that the centriolar structural components SAS-6 and SAS-4 are lost during late

embryogenesis, leaving splayed microtubules as a remnant of the centriole wall at the ciliary base.

The transition zone and axoneme are not completely formed at this time, indicating that cilia

maturation does not depend on the presence of intact centrioles. The hydrolethalus syndrome

protein HYLS-1 is the only known centriolar protein that continues to localize to the base of mature C.

elegans cilia. Loss of HYLS-1 severely impairs docking and entry of intraflagellar transport particles

and thus cilia assembly. Surprisingly, targeted degradation of HYLS-1 after initiation of ciliogenesis

does not appreciably affect ciliary structures. Taken together, our findings show that centrioles serve

as a structural template to initiate cilia formation but are dispensable for their maturation and

maintenance.

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Compartment-specific association of GABAB receptors and their effector ion

channels in cerebellar Purkinje cells

David Kleindienst(1), Rafael Luján(2), Carolina Aguado(2), Francisco Ciruela(3,4), Javier Cózar(2), Luis de la

Ossa(2), Kevin Wickman(5), Masahiko Watanabe(6), Yugo Fukazawa(7), Ryuichi Shigemoto(1)

Metabotropic GABA (GABAB) receptors mediate slow inhibition through their interaction with Gi/o protein and downstream effector molecules such as G protein-coupled inwardly-rectifying potassium

channels (GIRK) or voltage-gated calcium channels (Cav). This interaction is reported to be

compartment specific. Thus, in presynaptic terminals activation of GABAB receptors reduces

neurotransmitter release by inhibition of Cav [1], whereas in postsynaptic elements, it activates GIRK

channels, causing a hyperpolarizing outflow of K+ [2]. Here, we investigated the spatial relationship of

GABAB receptors with two of their effector molecules, Cav2.1 and GIRK2, in dendritic shafts and spines

of cerebellar Purkinje cells and in presynaptic active zones of parallel fibres in mouse cerebellum. To

this end, we used SDS-digested freeze-fracture replica labelling immunoelectron microscopy, a

sensitive method enabling quantitative high-resolution detection of membrane proteins in brain tissue

[3]. We conducted double labelling of GABAB1 with Cav2.1 or GIRK2 using immunogold particles of

distinct sizes. To assess whether these molecules are co-localized, we developed the Gold Particle

Detection and Quantification (GPDQ) software. GPDQ first semi-automatically detects gold particles in

a delineated area of the image and then carries out two types of simulation, random and fitted. Using

random simulation in GPDQ, we found that GABAB1, GIRK2 and Cav2.1 are all significantly clustered in

all compartments. Fitted simulation (Fig. 1) takes the original distribution of one kind of particles into

account and ensures that distribution of distances between the simulated particles of the molecule of

interest are not significantly different from that of distances between the corresponding real particles.

Comparison of Nearest Neighbour Distances (NNDs) from real or simulated GIRK2/Cav2.1 to real

GABAB1 particles can then reveal whether a significant association of each of these molecules to GABAB1

exists. We found significant associations of GABAB1 and GIRK2 in dendritic spines, but significant

dissociation in dendritic shafts. On the other hand, GABAB1 and Cav2.1 were selectively associated in

dendritic shafts. These results indicate compartment- and molecule-specific regulation of co-clustering

of GABAB1 and its effector molecules, which may support compartment-specific GABAB1 functions.

Figure 1: Example of a fitted simulation of GIRK2 in a dendritic spine. Scale bar: 100nm.

[1] Kaupmann, Klemens, et al. 1998, Proceedings of the National Academy of Sciences 95, no. 25, 14991–96. [2] Takahashi, Tomoyuki et al. 1998, Journal of Neuroscience 18, no. 9, 3138–46. [3] Masugi-Tokita, Miwako and Ryuichi Shigemoto. 2007, Current Opinion in Neurobiology 17, no. 3, 387–93.

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Characterization of the Vasa vasorum in the human great saphenous vein

by SEM and 3D-morphometry of vascular corrosion casts

Markus Herbst (1), Thomas Hölzenbein (2), Bernd Minnich (3)

(1) University of Salzburg, Physics Didactic Unit , Hellbrunnerstraße 34 , 5020 Salzburg (2) University Clinics of Vascular and Endovascular Surgery (PMU), Müllner Hauptstraße 48, 5020 Salzburg

(3) University of Salzburg, Vascular & Exercise Biology Unit, Hellbrunnerstraße 34 , 5020 Salzburg

"Vasa vasorum" (VV) derives from Latin and literally means "vessels of the vessels". Hence, the VV are

a network of small arterioles, venules and capillaries which supply the outer two layers of the wall

tissue of large blood vessels with oxygen and nutrients. The largest blood vessels in the body (e.g. the

human great saphenous vein, the aorta, etc.) depend on this supporting network to maintain their

health and function. Thus, the Vasa vasorum are an important part of the blood circulatory system.

In this study VV were studied in explanted segments of the human great saphenous vein (Vena saphena

magna, HGSV), taken during harvesting for coronary bypass grafts or extirpation of varicose vein

segments at the University Clinics for Vascular and Endovascular Surgery (PMU Salzburg), using

vascular corrosion casting (VCC), scanning electron microscopy (SEM, FEI/Philips XL-30 ESEM) and 3D-

morphometry (M3).

The aim of this study was the examination of the three-dimensional arrangement of the Vasa vasorum

in healthy and pathological (varicose) conditions. Moreover, it was intended to identify the most vital

segments of the HGSV in order to improve the results of bypass surgeries.

A meticulous analysis of the whole delicate microvascular system of the VV of the HGSV and its three-

dimensional arrangement (Fig. 1) is presented. It is one of the first studies yielding detailed quantitative

data on the geometry of the HGSV’s Vasa vasorum. Hence, a detailed insight into the optimality

principles (minimal lumen volume, minimal pumping power, minimal lumen surface and minimal

endothelial shear force) underlying the design of this microvascular network is given.

Arterial feeders originating from nearby arteries were

found to approach the HGSV every 15mm,

subsequentially forming a rich capillary network within

the adventitia and the outer two thirds of the media in

normal HGSV. In HGSV with intimal hyperplasia capillary

meshes of the VV were found to extend into the inner

layers of the media.

Measurements of spatial branching-off angles in

bifurcations and consecutive optimality calculations

showed that in both, the medial and distal part of the

HGSV, data are homogenously distributed close to the

theoretical optimum of vessel diameters.

Figure 1: The Vasa vasorum run predominantly parallel

to the longitudinal axis (LA) of the HGSV. Vessels having a longitudinal arrangement are defined as

orders 1 & 3. Orders 2 & 4 indicate vasa with a circular arrangement. Arterial vasa (A) are coloured in

red, venous vasa (V) in blue & capillaries (c) in orange. Arrows indicate the direction of blood flow.

[1] Herbst, M., Hölzenbein, T. & Minnich, B. (2014) Microscopy and Microanalysis, 20, 1120–1133.

17

The effects of double annealing on medium manganese steel

Tia Truglas(1), Christian Commenda(2), Martin Arndt(2), Daniel Krizan(2), Heiko Groiss (1, 3)

(1) CEST Competence Center for Electrochemical Surface Technology, Viktor Kaplan Str. 2, 2700 Wiener Neustadt

(2) voestalpine Stahl GmbH, voestalpine-Straße 3, 4031 Linz

(3) Zentrum für Oberflächen- und Nanoanalytik, Johannes Kepler Universität Linz, Altenberger Str. 69, 4040 Linz

The mechanical properties of batch or continuously annealed medium manganese steel grades with

an ultrafine-grained α + γ microstructure make them a promising candidate for the third generation of

advanced high strength steels. In the present study the effects of advanced continuous annealing

processes were investigated for a 0.1C6Mn2.2Al medium manganese steel with various electron

microscopy techniques, whereby a full microstructure analysis with EBSD and TEM was done after

single and double annealing. Compared to the former heat treatment the latter one contained an

additional annealing step using full austenitization and subsequent quenching prior to the final

intercritical annealing. EBSD phase maps revealed a fully α + γ microstructure with fine grain diameters

of around 400 nm for both steel types, whereas the simple annealed steel still contained larger ferrite

grains and a higher amount of low-angle grain boundaries (

18

Phase characterization in Ni-base superalloy Rene 65

Tomasz Wojcik(1), Markus Rath(1), Ernst Kozeschnik(1)

(1) Institute for Materials Science and Technology, TU Wien, Getreidemarkt 9, 1060 Wien, Austria

The Ni-base superalloy Rene 65 is a newly introduced cast and wrought alloy, used for turbine disk

applications in aero-engines. The fine-grained and γ’-strengthened alloy was developed to increase

service temperatures up to over 700°C and therefore, enhance the efficiency of aero-space turbines.

Additional, this cast and wrought alloy can be manufactured at lower costs than the conventional

powder metallurgy alloys.

In this work, the secondary phases occurring in the as-received material as well as in different heat

treatment conditions are characterized by transmission electron microscopy (TEM). In the as-received

material condition, a tri-modal size distribution of γ’ precipitates is found ranging from a few

nanometers up to approx. 5 µm. In addition, borides are found preferable at grain boundaries with a

size of approx. 1 µm. The boride phases could be identified by means of energy-dispersive X-ray

spectroscopy (EDX), electron energy loss spectrometry (EELS) with selected area electron diffraction

(SAED) as tetragonal M3B2 and M5B3, respectively, with Cr and Mo as the main metallic constituents.

For different cooling rates, a change in the morphology and size distribution of the γ’ precipitates is

found. These results are compared with thermo-kinetic precipitation simulations using the MatCalc

software package.

19

Viscoelastic stress relaxation of TiAl thin film under tension measured by

selected area electron diffraction

Christian Ebner(1), Rohit Sarkar(2), Jagannathan Rajagopalan(2), Christian Rentenberger(1)

(1) University of Vienna, Physics of Nanostructured Materials, Boltzmanngasse 5, 1090 Vienna, Austria

(2) Arizona State University, Department of Materials Science and Engineering, School for Engineering of Matter Transport and Energy, Tempe 85287, USA

Metallic glasses are a new class of materials with very distinct properties, making them promising

materials for structural applications [1]. Understanding the mechanisms of deformation and modelling

these on an atomic level is a challenge, which has to be overcome to fully take advantage of the

materials properties.

Here, we present our study of the time-dependent viscoelastic strain response of an amorphous TiAl

thin film to changes of the external stress. In-situ tensile tests are performed using a Phillips CM200

TEM operating at 200kV. Selected area electron diffraction (SAED) is used as a method to extract the

local atomic-level elastic strain [2]. Elliptic distortions of the radial intensity maxima positions of the

SAED patterns are introduced by tensile straining (cf. Fig. 1(a)). By precisely measuring these

distortions, the 2-dimensional strain tensor is calculated with respect to a reference pattern. This

allows to quantify the principal strain e1 (parallel) and e2 (perpendicular to the loading direction) as a

function of the external stress σ. The specimen is loaded and unloaded stepwise to different levels of

external stress, denoted as states 0-4. After each stress change a time series of SAED patterns with 2s

resolution is acquired. Patterns are recorded for times up to 1h. The changes in principal strain Δe1

with respect to the first pattern of the time series are shown in Fig. 1(b). Fitting of these changes with

a relaxation time model function gives a good fit only if two different relaxation times are used. In

addition, a dependence of the relaxation times τ on the stress step Δσ is observed.

Figure 1: (a) SAED pattern of TiAl: By applying a uniaxial tensile stress to the specimen, elliptic

distortions of the diffraction pattern arise as illustrated by the sketch. The peak shifts are used to track

the local atomic level elastic strain. (b) Changes of the strain over time: The time dependent strain

response of the tensile specimen is recorded for two loading and two unloading steps. The difference

in strain over a period up to 1h is extracted from the SAD patterns and fitted by a model function

consisting of two distinct relaxation times, to obtain the best fit.

[1] Greer, A.L. (2009) Materials Today, 12, 1-2, 14-22. [2] Ebner, C., Sarkar, R., Rajagopalan, J. & Rentenberger, C. (2016) Ultramicroscopy, 165, 51-58. C. E. and C. R. acknowledge financial support by the Austrian Science Fund FWF: [I1309]. R. S. and J. R. acknowledge funding from the National Science Foundation (NSF) grants CMMI 1400505 and DMR 1454109.

20

HRTEM study of Ca doped Bismuth Ferrite

Ulrich Haselmann (1), Yurii P. Ivanov (1) and Zaoli Zhang (1), (2)

(1) Erich Schmid Institute of Material Science, Jahnstraße 12, 8700 Leoben

(2) Montanuniversität Leoben, Franz-Josef-Straße 18, 8700 Leoben

Bismuth Iron Oxide (BiFeO3) has been attracting lots of scientific attention in the past years, especially

for being one of the few single phase multiferroic materials with magnetoelectric coupling at room

temperature, of whom it shows a very high antiferromagnetic Néel temperature (TN ≈ 370°C) and

ferroelectric Curie temperature (TC ≈ 830 °C). In particular, the electrical control of the

antiferromagnetic domains was successfully demonstrated. [1]

In bulk form BiFeO3 shows the rhombohedral space group R3c, but when deposited as thin film it can

take other forms due to epitaxial strain induced during the growth, as for example super-tetragonal

and distorted rhombohedral forms with LaAlO3 as substrate [2].

Here we present a structural characterization of BiFeO3 doped by Ca (Ca0.1Bi0.9FeO3) via transmission

electron microscopy using a JEOL 2100F equipped with CS-Corrector and operated at 200 keV. The

Ca0.1Bi0.9FeO3 was grown on a Strontium Titanium Oxide (SrTiO3) substrate (001) with a buffer layer of

Strontium Rubidium Oxide (SrRuO3) (001) used also as electrode for electrical characterization. For

TEM study cross-sections have been prepared by mechanical grinding and polishing and subsequent

ion milling. Some preliminary data will be shown.

[1] T. Zhao et al., “Electrical control of antiferromagnetic domains in multiferroic BiFeO3 films at room

temperature.,” Nat. Mater., vol. 5, no. 10, pp. 823–9, 2006.

[2] R. Huang et al., “Atomic-scale visualization of polarization pinning and relaxation at coherent

BiFeO3/LaAlO3 interfaces,” Adv. Funct. Mater., vol. 24, no. 6, pp. 793–799, 2014.

We kindly acknowledge the financial support by the Austrian Science Fund (FWF): No. P29148-N36.

Our gratitude also goes to the group of Prof. Yinghao Chu from the National Chiao Tung University in Taiwan for

providing the samples.

21

Introducing overlapping grain boundaries in chemical vapor deposited

hexagonal boron nitride monolayer films

Bernhard C. Bayer (1), Sabina Caneva (2), Timothy J. Pennycook (1), Jani Kotakoski (1),

Clemens Mangler (1), Stephan Hofmann (2), Jannik C. Meyer (1)

(1) University of Vienna, Faculty of Physics, A-1090 Vienna, Austria

(2) University of Cambridge, Department of Engineering, CB3 0FA, Cambridge, UK

Hexagonal boron nitride (h-BN) is a two-dimensional (2D) insulator with a wide application profile,

including its use as an ultimately thin dielectric in electronics, a tunnel barrier in spintronics, an

encapsulation- and barrier-layer in electronics and metallurgy and a suspended separation membrane

in nanofluidics. A key technological challenge is the scalable manufacture of h-BN, in particular as a

continuous film of controlled layer number and high crystalline quality. Catalytic chemical vapor

deposition (CVD) has emerged as a promising technique to achieve growth of such h-BN films, including

exclusive monolayer growth [1-3]. CVD h-BN films are typically poly-crystalline and control of their

microstructure, in particular grain boundary (GB) structure, is important for many applications. Current

literature reports the nature of as-grown GBs in h-BN as atomically stitched, composed of defect lines

within a h-BN monolayer. Preferential pinhole formation is reported at such atomically stitched GBs,

and due to their structure they are intrinsically prone to electrical breakdown, chemical attack or

mechanical failure, all of which may render monolayer h-BN films ineffective in their envisaged

applications.

Here we show using complementary (scanning) transmission electron microscopy ((S)TEM) techniques

that GBs in monolayer h-BN films grown by scalable catalytic CVD can not only be atomically stitched

but can also be overlapping in nature. We show that in these overlapping GBs two h-BN monolayer

grains merge via the self-sealing formation of a turbostratic bilayer region of limited width and thereby

without formation of a defect line within the monolayer. We characterize this overlapping GB structure

in detail, identify catalytic CVD conditions that result in such GB structure and propose possible

underlying catalytic growth mechanisms. Our data suggests that overlapping GBs are comparatively

resilient against detrimental pinhole formation, as evolving defects in one layer are sealed by the

second layer. Thus overlapping GBs may be technologically advantageous for the many h-BN

applications for which continuous pinhole-free h-BN monolayers are key.

[1] Chem. Mater., 26, 6380 (2014).

[2] Nano Lett., 15, 1867 (2015).

[3] Nano Lett., 16, 1250 (2016).

We kindly acknowledge financial support from the European Union’s Horizon 2020 research and innovation

program under the Marie Skłodowska-Curie Grant Agreement 656214-2DInterFOX (B.C.B.) and from the Austrian

Science Fund (FWF, P25721-N20).

22

Combined analytical TEM and magnetic investigation of the effects of neutron irradiation on Nb3Sn superconductors

Pfeiffer Stephan(1), Bernardi Johannes(1), Stöger-Pollach Michael(1), Baumgartner Thomas(2), Eisterer Michael(2), Ballarino Amalia(3)

USTEM, TU Wien, Wiedner Hauptstraße 8-10, 1040 Vienna, Austria

(2) Atominstitut, TU Wien, Stadionallee 2, 1020 Vienna, Austria

(3) CERN, 1211 Geneva 23, Switzerland

An increase of the high field critical currents in commercial Nb3Sn wires by about 50 % is required for

the design of FCC-hh (Future Circular Collider study for hadron/hadron collisions) superconducting

magnets. A previous study touched already this ambitious goal by producing additional pinning

centers. They were created by inducing crystal defects in the superconducting material by means of

fast neutron irradiation. In the present study, the underlying mechanisms are investigated through

combined microstructural and magnetic analysis. This knowledge will be important for industrial

manufacturing of the required high-performance superconducting cables.

The nuclear research reactor of TU Wien was used to irradiate Nb3Sn wires and already prepared

TEM (transmission electron microscopy) specimens. Micro- and nanostructural examinations of grain

geometry, grain boundary morphology, compositional gradients, local texture and defect structure

were performed in the TEM before and after irradiation by employing high-resolution TEM, EDX

(energy-dispersive X-ray spectroscopy), EELS (electron energy loss spectroscopy) and selected area

diffraction.

The results thereof are correlated with measurements of the superconducting properties, in

particular scanning Hall probe experiments and SQUID magnetometry to determine the global critical

current as well as the local critical current density within the superconducting subelements.

This study contributes to a better understanding of the influence of irradiation damage and the

resulting microstructure on local superconducting properties and ultimately on the macroscopic

performance of the superconductor.

Figure 1: High resolution image of neutron impact site (left) and remanent field Hall scan (right).

23

Advanced characterization of materials using atomic resolution TEM

Zaoli Zhang

Erich Schmid Institute of Materials Science, Austrian Academy of Sciences, Leoben, 8700, Austria

In this presentation, some recent results acquired using our aberration corrected TEM will be shown.

The first part will focus on bulk nanostructured materials prepared by severe plastic deformation.

Here, the evolution of the structural and chemical composition in the nanostructured materials with

temperature was tracked in real-time via simultaneous in-situ imaging and spectroscopy analysis. It

demonstrates that the nanostructured materials are not only subjected to a structural change but also

to an obvious chemical composition fluctuation upon annealing. Real-time imaging and composition

determination allow further analyzing the dynamic behavior in nanocrystalline materials in details, i.e.

deducing the instantaneous diffusion coefficients and excess vacancy concentration generated by

deformation.

The second example will be on the CrN/AlN multilayers. CrN/AlN multilayers exhibit a peak in hardness

of ～40 GPa under certain bilayer period (Λ). These improvements in mechanical properties in

comparison with their monolithic counterparts have a close relationship with the existence of a

metastable face-centered cubic (fcc) AlN phase which can be epitaxially stabilized in thin films. Here,

interplanar spacing oscillations in cubic CrN/AlN multilayers were experimentally observed by using

spherical aberration-corrected high-resolution transmission electron microscopy (HRTEM), and were

corroborated by first principles calculations. Electron spectroscopy and microscopy were employed to

analyze the strain distribution in the multilayers and obtain generalized relationships between the

electronic structure on the one hand, and (non-)stoichiometry or strains in the multilayers on the other

hand. The present study provides atomic-scale insights in the mechanisms of extraordinary strength

pertaining to the CrN/AlN multilayers.

I would like to thanks to Reinhard Pippan, Jinming Guo, Julian Rosalie and Xunlong Gu (at the Erich

Schmid institute), Matthias Bartosik and Paul H.Mayrhofer (TU Wien), David Holec, Rostislav Daniel

and Christian Mitterer (Montanuniversität Leoben) for discussions and depositing the film materials.

24

High Resolution Visualisation of Iron Deposits in the Human Brain in Health

and Disease

Mariella Sele(1), Christoph Birkl (2), Stefan Ropele (2), Johannes Haybäck (3), Walter Gössler (4) and Gerd

Leitinger (1)

(1) Medical University of Graz, Institute of Cell Biology, Histology and Embryology, Harrachgasse 21, 8010 Graz (2) Medical University of Graz, Division of General Neurology, Auenbruggerplatz 22, 8036 Graz

(3) Medical University of Graz, Institute of Pathology, Auenbruggerplatz 25, 8036 Graz (4) University of Graz, Institute of Chemistry, Universitätsplatz 1, 8010 Graz

During aging from birth until the fourth decade of life, iron accumulates in various areas of the brain to different degrees [1]. Until now little is known how and why some brain areas contain significantly more iron than others. Moreover, iron accumulations are associated with many inflammatory and neurodegenerative diseases like Alzheimer’s disease (AD) or multiple sclerosis [2, 3]. Dysregulation of iron homeostasis or its release from damaged tissue can induce the production of radical oxygen species, cause oxidative stress and consequently apoptosis. It is known that Iron in the brain is mostly stored in glial cells or neurons [4] but how it gets there or why is it so unequally distributed in the brain is not known. Therefor we aim to elucidate the distribution, size and composition properties of the iron - containing ferritin in the human brain. With our comprehensive approach we combine findings from quantitative magnetic resonance imaging, mass spectrometry, analytical electron microscopy and immunochemical tests. In the analytical EM part we use energy filtered transmission electron microscopy (EFTEM) and Energy-dispersive X-ray spectroscopy (EDX). This interdisciplinary course of action will enable us to investigate which cell types and which subcellular compartments act as iron stores in the human brain. We thus aim to elucidate the mechanism that accumulates iron in the brain. Preliminary results confirm that the cellular and subcellular distribution of ferritin iron differs between areas with high and low iron content in human brain samples. The basal ganglia which consist of the globus pallidus (205 ± 32 ppm Iron) and the putamen (153 ± 29 ppm Iron) is the brain area in which the most iron was found [5]. In these areas were are able to show clusters of iron-loaded particles within oligodendrocytes. Our aim is to further characterise these Iron/Ferritin clusters. We propose that iron deregulation must be detectable in Alzheimer’s disease patient’s samples when studying the iron distribution of the basal ganglia. A better understanding of the iron distribution and the iron metabolism in the human brain could open new possibilities in treatment of neurodegenerative diseases.

[1] Hallgren, B. & Sourander P. (1958) Journal of Neurochemistry, 3, 41-51. [2] Smith, M., Harris P., Pauly, S., Sayre, L., & Perry G. (1997) Proc Natl Acad Sci U S A., 94, 9866-9868. [3] Khalil, M., Teunissen, C. & Langkammer, C. (2011) Mult Scler Int, 2011, 6 [4] Merugo, R., Asano, Y., Odagiri, S., Li, C. & Shoumura, K. (2008) Arch Histol Cytol, 71, 205-222 [5] Langkammer C., et al. (2010) Radiology, 257, 2, 455-462

We kindly acknowledge financial support by the Austrian Science Fund (FWF):[P-29370B27]

25

Impact of fibrinogen concentration on blood clot formation

Christoph Dibiasi1, Leon Ploszczanski2, Helga Lichtenegger2, Ursula Windberger1

(1) Department of Biomedical Research, Medical University of Vienna, Vienna, Austria

(2) Institute of Physics and Material Science, University of Natural Resources and Life Sciences Vienna

Blood is a fluid organ composed of cells embedded in blood plasma. During coagulation blood changes

it’s state to a solid by interlinking fibrin monomers to form a complex polymer attached to the cellular

components. This process can be quantified by measuring the viscoelastic properties of the clot, i.e.

shear storage modulus G’ by rheometry.

The aim of this study was to measure the impact of fibrinogen concentration (the precursor protein of

fibrin) on clot forming kinetics and final clot composition. For each measurement, 0.58mL blood of

human volunteers (n = 8) was sheared in the plate-plate measurement geometry of the rheometer

Physica MCR 301 (Anton Paar, Graz, Austria) until establishment of a G’ plateau, at which point the

blood clot was removed and fixated in formaldehyde. After drying and coating with Au the specimens

have been examined at high vacuum conditions and 20kV in a FEI 250 FEG ESEM.

We had defined two sample groups: One with normal fibrinogen concentration (mean 232.20 ± 50.75

mg/dL) and one with fibrinogen added to a final concentration of 1010.00 ± 196.83 mg/dL. SEM

pictures of clots from both groups are shown in fig 1 and 2. The fibrin network shows a greater density

with more fibers and less voids in the network. This corresponds to a higher G’ (223.50 ± 59.68 Pa in

the baseline group vs. 327.88 ± 58.86 Pa).

Fig 1: Clot with normal fibrinogen concentration

Fig 2: Clot with high fibrinogen concentration

26

Using electron microscopy as a method to monitor autophagy

Hubert Virginie1, Langer Brigitte1, Rees Andrew1, and Kain Renate1.

1Institute of Clinical Pathology, Medical University of Vienna

Autophagy is an evolutionary process used to eliminate cytoplasmic material through its accumulation

into a sealed structure the autophagosome that will then fuse with a lysosom. In the last few years,

major improvements have been made in the methods applied to monitor this process; among them is

transmission electron microscopy (TEM), which presents a much higher resolution than, for instance,

indirect immunofluorescence detection of antigens and subcellular structures. Following embedding

in epoxy resins, the autophagic compartments can easily be identified based on morphologic features;

autophagosomes are large structures often localized close to the endoplasmic reticulum, surrounded

by a double membrane and containing cytoplasmic material while lysosomes present a spherical shape

filled with electron dense material. Moreover, quantifying methods allow to gain information into the

nature of of proteins and (degraded) organelles accumulating within the cells and the subcellular

compartments. They thus allow to monitor the process of organelle trafficking and fusion in detail and

to investigate pathological processes, like blockage of fusion. Using TEM to study autophagy in

fibroblast cell lines sufficient and deficient for the lysosomal associated membrane protein-2 (LAMP-

2), we successfully demonstrated a new role of LAMP-2 in the fusion of the autophagosome with the

lysosomes. Moreover, we could also identify the presence of intact lysosome-like vesicles in the

autophagosomes of LAMP-2 deficient cells, a phenomenon previously unreported and identifiable only

by electron microscopy. This example successfully demonstrated the necessity of TEM to study

autophagy and the need to develop new methods by combining them with other techniques such as

immunofluorescence.

27

Serum derived exosomes as a putative diagnostic tool for ANCA associated

vasculitis

Stefan M. Schulz(1), Dario A. Leone(1), Helga Schachner(1), Andrew J. Rees(1), Renate Kain(1)

(1) Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria

Exosomes are extracellular vesicles present in most bodily fluids that recently came into the focus of

research for their unique properties and their wide range of potential applications in diagnosis and

treatment.

Exosomes are enclosed by a double membrane, secreted by most cell types and contain proteins,

DNA and RNA. They originate from various cell types and their distinct molecular signatures could

potentially constitute a novel diagnostic tool in autoimmune disease and cancer. While exosome

isolation from cell culture supernatants is well established, isolation from serum or plasma remains

challenging.

The aim of this project is to isolate individual populations of exosomes from human serum, to

identify their different cellular origin and determine whether their molecular composition is modified

in ANCA associated vasculitis (AAV), an autoimmune disease characterised by inflammation of small

blood vessels. We established reliable and robust protocols to isolate exosomes from serum or

plasma of AAV patients and healthy controls that are used to identify proteins expressed on the

surface of exosomes that could serve as disease specific biomarkers.

The purity of exosomes isolated from serum by ultracentrifugation and/or commercially available

reagents was assessed and their size and morphology validated using transmission electron

microscopy (TEM). Localization of membrane proteins was confirmed using immunogold labelling.

Sample protein content was evaluated using 1D-SDS-PAGE and Coomassie staining. Exosome

markers, e.g. the tetraspanins CD9 and CD63, were used to confirm the presence of exosomes in

Western Blot assays and real-time as well as QPCR were used to analyse their RNA/DNA content.

Our early results show, in accordance with previous publications [1], that ultracentrifugation resulted

in exosome populations with a larger diameter and achieved far lower protein yields than

commercial kits that are based on solubility that produced highly concentrated samples with a wide

particle size range.

[1] Helwa I, Cai J, Drewry MD, Zimmerman A, Dinkins MB, Khaled ML, et al. (2017) PLoS ONE 12(1): e0170628.

doi:10.1371/journal.pone.0170628

28

Flash and Freeze: combining high-pressure freezing and optogenetics to

evaluate synaptic transmission

Carolina Borges-Merjane, Olena Kim, Peter Jonas

Institute of Science and Technology (IST) Austria, Am Campus 1, 3400 Klosterneuburg

The visualization of the ultrastructure of synapses by electron microscopy with high pressure

freezing (HPF) has enabled a better understanding of their morphological properties and subcellular

structures. However, synaptic transmission is a dynamic process, and HPF alone only captures static

images. The recently developed “Flash and Freeze” technique1,2,3 combines optogenetics with HPF

allowing for the visualization of action potential evoked membrane dynamic changes at synapses

during neurotransmission. With this powerful technique, a brief pulse of light activates the genetically

expressed light-activated channel channelrhodopsin in targeted cells, leading to action potential

initiation and inducing vesicle fusion to the membrane. After light stimulation the tissue is frozen by

HPF with a pre-set timed delay after onset of stimulus, thus allowing for capture of events at different

time points after synaptic transmission onset, from exo- to endocytosis.

We are using the Leica ICE with Light Stimulation system4 for “Flash and Freeze”, to assess

mechanisms underlying synaptic transmission at the mossy fiber-to-CA3 pyramidal cell synapse - in

mouse hippocampus during basal transmission and after short-term plasticity. We use acute brain

slices from 21 to 28 day-old mice and organotypic slice culture, prepared from 5 to 7 day-old mice,

maximum two weeks old. We are using transgenic mice, specifically with cre-recombinaseER in dentate

gyrus granule cells under the prox1 gene promoter, and are currently testing crosses with reporter

lines expressing the light-activated channel channelrhodopsin for specific expression. With this

method and approach, we hope to have a better understanding of presynaptic changes that occur at

the mossy fiber terminals, contacting CA3 pyramidal neurons in hippocampus. Figure 1: Left: Confocal z-stack (0.5 μm steps) of a horizontal section of hippocampus from an Ai27 het/Prox1-

creER hem mouse injected with tamoxifen. Neurons labeled with anti-NeuN antibody (cyan) and dentate gyrus

(DG) granule cells labeled in red showing specificity of expression of channelrhodopsin. Scale 200 μm. Middle:

Voltage-clamp recording of a granule cell from an acute brain slice of a transgenic mouse showing response to 5

ms blue light stimulation. Overlaid traces showing multiple trials. Right: Transmission electron microscope image

of a DAB stained, fixed hippocampal slice after pre-embedding with anti-RFP antibody (in this mouse TdTomato

is fused with channerhodopsin). MFs: mossy fibers; MFB: mossy fiber bouton. Scale 1 μm.

[1] Watanabe, S., Liu, Q., Davis, M.W., Hollopeter, G., Thomas, N., Jorgensen, N.B. & Jorgensen E.M. (2013) Elife.

Sep 3;2:e00723. doi: 10.7554/eLife.00723.

[2] Watanabe, S., Rost, B.R., Camacho-Pérez, M., Davis, M.W., Söhl-Kielczynski, B., Rosenmund, C., & Jorgensen,

E.M. (2013) Nature, 504(7479):242-7. doi: 10.1038/nature12809.

[3] Watanabe, S., Davis, M.W., & Jorgensen, E.M. (2014) Nanoscale Imaging of Synapses, Chapter 3, 43-57

[4] Leica Microsystems, Vienna, Austria. http://www.leica-microsystems.com/products/sample-preparation-for-electron-microscopy/cryo-preparation-systems/details/product/leica-em-ice We thank our funding sources: C.B.M MSCA H2020 708497; PJ (OK) FWF W1205-B09; PJ ERC 692692

29

SEM on agarose-based chromatographic beads – how to recalculate a reference SAXS scattering signal from an image

Jacek Plewka1,2, Leon Ploszczanski1, Heinz Rossbacher1, Rupert Tscheliessnig2, Alois Jungbauer2, Harald Rennhofer1, Helga Lichtenegger1

(1) Institute of Physics and Material Science, University of Natural Resources and Life Sciences Vienna (2) Austrian Centre of Industrial Biotechnology, ACIB GmbH

With the total sales of $75 billion, monoclonal antibodies are the most lucrative product on bio-

pharmaceutical market accounting for over 50% of worldwide market. Agarose-based chromatography

media, used for protein-A affinity chromatography - method for antibody capturing, are then of the

upmost importance in biopharmaceutical industry. Being the most expensive steps in antibody

purification process its full understanding, including the mechanical properties on nanometer scale, is

essential to ensure the performance.

Here, we would like to demonstrate a method for inner structure visualization of agarose-based

chromatographic beads using Scanning Electron Microscopy (SEM) approach and subsequent image

processing to reconstruct Small Angle X-Ray Scattering (SAXS) images recorded on the same material.

Although, those two methods provide quite different approaches (microscopy gives local details on

surface, whereas SAXS provides global parameters), they are often employed together for deeper

understanding of analyzed materials on the nanoscale. However, traditionally SEM is only used for

morphological examination of specimen, whereas we propose to use it for further processing to get a

reference signal for SAXS method as well.

In the Figure below a short summary of used methods is shown. A dehydrated resin is embedded in LR

white resin and sliced using microtome to ensure smooth surface of the specimen and then coated

with thin layer of gold prior SEM image capturing. Captured images of sufficiently good resolution in

nanometer scale are then further processed to obtain a reference SAXS signal using 2D image fast

Fourier transform (FFT) and subsequent 1D radial averaging of the image in reciprocal space.

Alternatively, one can also binarize the image and randomly probe the surface of a bead with

significant number of points to calculate the Pair Distribution Function out of them (measure of

distances and their probabilities), which using the Debye formula can be recalculated to yield the SAXS

reference signal as well. Such a reference signal can be used to check the background subtraction

quality for SAXS method or to extrapolate the SAXS signal to the very low-q regime (corresponding to

a size range larger than 100 nm), where due to experimental restrictions no SAXS information is

available.

30

Babinet principle for plasmonic antennas: complementarity and differences

Michal Horák (1), Vlastimil Křápek (1,2), Martin Hrtoň (1,2), Michael Stöger-Pollach (3),

Tomáš Šamořil (1,2), Filip Ligmajer (1,2), Tomáš Šikola (1,2)

(1) Central European Institute of Technology, Brno University of Technology, Purkyňova 123, 612 00 Brno, Czech Republic

(2) Institute of Physical Engineering, Brno University of Technology, Technická 2, 616 69 Brno, Czech Republic

(3) University Service Center for Transmission Electron Microscopy, Vienna University of Technology, Wiedner Hauptstrasse 8-10, 1040 Vienna, Austria

Babinet principle relates the optical response of apertures in thin films and their complementary

analogues – solid barriers or particles. Originating in the wave theory of light and analysis of diffraction,

it has recently entered the field of plasmonics. According to Babinet principle, localized surface

plasmons in complementary particles and apertures have identical resonance energies and their near

field are closely linked: The electric field distribution of a specific in-plane polarization for an aperture

corresponds to the magnetic field distribution of perpendicular polarization for a particle [1]. On the

other hand, additional differences can be related to different fabrication processes and experimental

techniques involved in the characterization of real structures.

To assess the theoretically predicted Babinet complementarity, we have studied a set of gold disc-

shaped plasmonic antennas with various diameters, both particles and apertures. Plasmonic antennas

were fabricated by focused-ion-beam lithography of thin gold layer on silicon nitride membrane.

Localized plasmon resonances were characterized by cathodoluminescence and electron energy loss

spectroscopy (EELS). Babinet complementarity was confirmed for main plasmon properties such as

resonance energies, but differences were found, for example, for the excitation efficiency (Fig. 1).

Figure 1: Left: Cathodoluminescence spectra of gold dics-shaped plasmonic particles and apertures of

various diameters. The excitation electron beam was focused on the edge of the structure to maximize

the excitation efficiency. Note generally stronger response of the apertures. Right: Dispersion relation

of localized plasmon resonances. Peak energy of cathodoluminescence is shown as a function of

reciprocal value of the antenna diameter (resembling the wave number). Experimental values show

no difference between particles and apertures and closely follow the values obtained from numerical

simulations.

[1] Hentschel, M., Weiss, T., Bagheri, S., & Giessen, H. (2013) Nano Letters, 13, 4428–4433.

We kindly acknowledge financial support by Czech Science Foundation, project No. 17-25799S.

31

Hybrid plasmonics: From plasmon-plasmon to plasmon-exciton coupling

Franz-Philipp Schmidt(1,2), Harald Ditlbacher(1), Andreas Hohenau(1), Ulrich Hohenester(1), Ferdinand

Hofer(2), and Joachim R. Krenn(1)

(1) Institute of Physics, University of Graz (2) Institute for Electron Microscopy and Nanoanalysis, Graz University of Technology

The coupling of plasmonic nanoparticles can lead to extremely localized fields and is thus a central

topic in nanooptics research and application. In our work, we show that the spectral response of a

single rectangular plasmonic nanoparticle can be interpreted due to coupling of edge excitations,

leading to bonding and antibonding modes (Fig.1a,b) [1]. On one hand, we rely on high-resolution

experimental data from electron energy-loss spectroscopy, applied to a single lithographically

prepared silver cuboid. On the other hand, we use numerical simulations by the boundary element

method, finding excellent agreement with the experiment.

Going one step further we couple metallic with semiconducting nanostructures (Fig.1c) in terms of

plasmon-exciton coupling [2]. The importance of high energy resolution to differentiate subtle energy

shifts and splittings is demonstrated using a monochromated system in combination with advanced

data post processing routines (Fig.1d) [3].

Figure 1:

Plasmon-plasmon coupling: (a) EEL spectra extracted from three different regions of a silver

nanocuboid as indicated in the inset. (b) Simulated charge distribution of the dipolar plasmon edge

mode along a 300 nm long silver edge, which splits up into a bonding and antibonding mode due to

coupling of the opposite edge plasmons.

Plasmon-exciton coupling: (c) “Monochromated” HAADF images of a silver dimer and CdSe/ZnS

quantum dots and (d) corresponding EEL spectra in the gap region before (blue) and after (red) data

post processing.

[1] Schmidt, F.-P., Ditlbacher, H., Hohenau, A., Hohenester, U., Hofer, F., Krenn, J. R. (2016) Nano Letters, 16, 5152–5155. [2] Wei, J., Jiang, N., Xu, J., Bai, X., Liu, J. (2015) Nano Letters, 15, 5926–5931. [3] Schmidt, F.-P., Hofer, F., Krenn, J. R. (2017) Micron, 93, 43–51. This research was supported by the Austrian Science Fund FWF (P21800-N20, SFB F49), NAWI Graz and the Graz Center for Electron Microscopy.

32

Influences of the CMR effect on dielectric properties

Wolfgang Wallisch (1), Michael Stöger-Pollach (1), Edvinas Navickas (2)

(1) Technische Universität Wien, University Service Centre for TEM, Wiedner Hauptstrasse 8-10, 1040 Vienna (2) Technische Universität Wien, Institute of Chemical Technologies and Analytics, Getreidemarkt 9, 1040 Vienna

Over the last decades, double perovskite oxides are attractive materials due to their complex magnetic

and electrical behaviour. Their promising physical and electronic properties are interesting for a wide

range of applications. Among these properties, also the large negative magnetoresistance of

La2CoMnO6 (LCM) [1], which is known as the colossal magnetoresistance (CMR), is of great interest.

This effect is an metal-insulator transition describing the change of the resistance in the presence of a

magnetic field.

These days, transmission electron microscopy (TEM) equipped with energy filters are powerful tools

and its main advantage for probing band gaps is the high spatial resolution. It offers an opportunity to

investigate the influences of the physical consequences of the CMR effect on the electron energy loss

spectrometry (EELS) signal in TEM. The observation and detection of the change of the band structure

in the low energy range and the magnetic behaviour of the material with chemical sensitivity [2] by

using energy loss magnetic chiral dichroism (EMCD) will be presented.

The dielectric response is contained in the low loss spectrum, which is exhibited in Fig. 1. The 40 keV

and 200 keV spectra are shown at a temperature of 85 K in Fig. 1A. It is obvious that there is a difference

in the energy loss range of 1.5 eV to 4 keV. On the other hand, concerning the comparison of the 40

keV valence EELS (VEELS) spectra at different temperatures (Fig. 1B), an intensity variation is caused

by the CMR effect and not by the Čerenkov effect. The EMCD investigations are performed at 200 keV.

The CMR effect causes a magnetisability in a magnetic field of less than approximately 0.5 T [1]. The

chemical sensitivity of EMCD is shown in Fig. 1C, the EMCD effect can be observed at the Co edge in

the 85 K experiment.

Figure 1: (A) Unprocessed VEELS spectra recorded at 85 K at a sample thickness of 0.3 λ using 40 keV

and 200 keV, respectively. (B) Low loss spectrum recorded at room temperature (RT) and at 85 K using

40 keV electrons. The insertion shows the divergences between the RT and the 85 K spectrum. (C)

Normalized EELS spectrum of the LCM layer. The Co edge shows induced chiral electronic transitions

at 85 K.

[1] Mahato, R. N., Sethupathi, K. & Sankaranarayanan, V. (2010) Journal of Applied Physics, 107, 09D714. [2] Ennen, I., Löffler, S., Kübel, C., Wang, D., Auge, A., Hütten, A. & Schattschneider, P. (2012) Journal

of Magnetism and Magnetic Materials, 324, 2723-2726. The authors kindly acknowledge financial support by the Austrian Science Fund (FWF):[F4501-N16, F4509-N16].

33

Reaction of Ni and C thin films studied by TEM and SEM

Semir Tulić (1), Viera Skákalová (1), Thomas Waitz (1), Gerlinde Habler (2), Marián Varga (3), Alexander

Kromka (3), Viliam Vretenár (4), Mária Čaplovičová (4)

(1) Faculty of Physics, University of Vienna, Boltzmanngasse 5, 1090 Vienna, Austria (2) Department of Lithospheric Research, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria

(3) Institute of Physics, Czech Academy of Sciences, Cukrovarnická 10, Prague 6, Czech Republic (4) Slovak University of Technology, Center for Nanodiagnostics, Vazovova 5, 812 43 Bratislava, Slovakia

This work focuses on the catalytic reaction of thin Ni films (thickness 20 and 500 nm) with

nanocrystalline diamond (NCD; grains ~200 nm in diameter). The films are deposited on Si substrates

by magnetron sputter deposition to yield a Ni-NCD-Si sequence. After reaction by annealing at a

temperature of 900 °C, samples are studied prior and after the removal of any residual Ni surface layer

by using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). TEM

specimens were prepared in a cross-sectional geometry by focused ion beam (FIB) thinning using

protective layers of Au and Pt. After annealing of the samples with 500 nm thick Ni films (denoted NCD-

500), columns of NiSi2 are covered with a surface layer of C (see Figs. 1(a) and (b)). While graphite is

observed directly at the interface with NiSi2, with increasing distance from this interface, the layered

structure of graphite seems to become more disordered, finally yielding amorphous C (a-C) (see Fig.

1(c)). Graphite is arising by a catalytic reaction of the Ni with diamond [1]. NiSi2 is arising by a reaction

of Ni diffusing along the grain boundaries of the NCD towards the Si substrate. In addition, Si and C

have interacted to form nanocrystallites of SiC. After annealing of the samples with 20 nm thick Ni

films (denoted NCD-20), isolated Ni nanoparticles arise by dewetting of the Ni film; catalytic etching

by the Ni nanoparticles causes the formation of grooves in the NCD (see Fig. 2(a)) On top of the NCD,

a continuous layer of a-C is observed (see Figs. 2(b) and (c)). Since the catalytic reaction of Ni and C is

expected to yield the formation of graphite [1], in the present case the a-C might have formed by

radiation damage during the specimen preparation via FIB [2].

Figure 1: NCD-500. (a) SEM image of NiSi2 columns decorated with Ni nanoparticles. (b) TEM images

of (a) C-NiSi2-SiC-Si reaction nanostructures and (c) graphite gradually changing to amorphous C.

Figure 2: NCD-20. (a) SEM image of NCD showing grooves. TEM images (b) of the reaction layers and

(c) the interface between the NCD and a-C.

[1] H. Mehedi, et al., Carbon, 59, 448-456, 2013.

[2] R. Colby, et al., Diamond and Related Materials, 19 (2), 143-146, 2013.

We kindly acknowledge financial support by the Austrian Science Fund (FWF), Czech Science Foundation GACR

and Slovak Scientific Grant Agency VEGA:[AI0234421, 16-34856L, 1/1004/15].

34

Analytical FIB-SEM Tomography without Compromises

Fabián Pérez-Willard (2), Giuseppe Pavia (2), Wolfgang Schwinger(1)

(1) Carl Zeiss GmbH, Laxenburger Str. 2, AT-1100 Vienna, Austria

(2) Carl Zeiss Microscopy GmbH, Carl-Zeiss-Str. 22, DE-73447 Oberkochen, Germany

In materials research the capability to analyse comprehensively the microstructure of a specimen in

three-dimensions is becoming increasingly important. In this context X-ray and FIB-SEM microscopy –

the focus of this work – play a key role as they enable researchers to understand structural changes

caused by processing or use of a material across different relevant length scales [1]. As a result,

materials with better properties and performance can be developed more efficiently.

While FIB-SEM tomography provides its best spatial resolution of a few nm voxel size, when performed

at low accelerating voltages, usually between 1 and 2 kV, energy dispersive spectroscopy (EDS)

requires at least a factor of two to three larger landing energies for the excitation of the characteristic

EDS fingerprint. In the past, analytical FIB-SEM tomography was always performed at the lowest

acceleration voltage still compatible with the EDS analysis, thus sacrificing spatial resolution in SEM

imaging.

Recently, a software solution within the Atlas 5 tomography environment has been developed, which

allows automatic switching between two different sets of SEM conditions: A first one at low voltage

and current for the acquisition of high-resolution electron images and smallest possible voxel sizes.

And a second at a much higher voltage and current, for high-throughput EDS mapping with lower

spatial resolution and larger voxel sizes [2].

In this contribution, we will present some materials science examples to illustrate the advantages of

this new approach.

Figure 1: Exemplary slice from a FIB-SEM tomography dataset on a lead free solder sample (courtesy

of M. Cantoni, EPFL Lausanne). Electron imaging (left) was done at 1.8 kV using Inlens SE detection

with a voxel size of (10 x 10 x 10) nm³. The EDS maps were acquired every tenth slice at 6 kV with a

voxel size of (40 x 40 x 100) nm³.

[1] Merkle, A. et al. (2014) Microscopy and Analysis, 28(7), 10-13.

[2] Cantoni, M. et al. (2016) Proceedings of the 16th European Microscopy Congress, Lyon, France.

35

Dealing with light refraction in 3D mapping in combined Raman/SEM

Robert Striemitzer (1,2), Peter Pölt (1,2) ,Harald Fitzek (1),

(1) Institute of Electron Microscopy and Nanoanalysis, Graz University of Technology, NAWI Graz,

Steyrergasse 17, 8010 Graz

(2) Graz Centre for Electron microscopy, Steyrergasse 17, 8010 Graz

In recent years Raman-microscopy and scanning electron microscopy (SEM) were integrated in one

instrument, and the advantages of correlative Raman-microscopy and SEM could be demonstrated

[Zitat Raman/SEM Paper]. One advantage of a combined Raman/SEM is that depth profiling and 3D-

maps are possible using confocal Raman microscopy. Unfortunately, the sample has to be immersed

in oil for confocal microscopy as otherwise the refraction of light at the sample surface will lead to a

quick deterioration of the depth resolution with focus depth and a compressed depth scale [Evrall

1,2]. While it is possible to mathematically correct the compression of the depth scale, the lost

resolution cannot be recovered. Therefore, we are trying to make immersion possible in the vacuum

of a SEM chamber.

A measurement with the least information loss would occur with light not changing medium at all or

a change to media with the same refractive index (Reflection is negligible in both cases). To get such a

case usually oil with the preferred refractive index is dropped on the sample and an oil immersion

objective is used. Our approach is to introduce a thin film with a refractive index close to the

immersion oil between the sample and the oil. If the contact between the sample and the film is

satisfactory this would allow for immersion to be used in vacuum. The first experiments using

different methods are displayed in Fig. 1 and compared to the standard oil approach and measuring

at ambient air.

Figure 1: Visualisation of polymer multilayers (PS, PMMA, PS, PMMA) on a PET substrate measured

with different techniques: Space between sample and objective filled with a) Oil, b) Air, c) Tape and

Oil, d) Silicone Layer and Oil

The thickness obtained by measuring with oil corresponds with the known thickness of 52.5 µm. The

closest condition to vacuum (n = 1) is air (n 1.0001) and its result is compressed as expected. The

results coming from measurements with a protective adhesive tape and a silicone layer are within

uncertainty range equivalent to the measurement with oil.

[1] Worobiec, A., Potgieter-Vermaak, S., Brooker, A., Darchuk, L., Stefaniak, E., & Van Grieken, R. (2010),

Interfaced SEM/EDX and micro-Raman Spectrometry for characterisation of heterogeneous environmental

particles— fundamental and practical challenges, Microchemical Journal, 94(1), 65-72 [2] Evrall, N. (2004), Depth profiling with confocal Raman microscopy, Part I. Spectroscopy-Springfield then Eugene then Duluth, 19, 22-33 [3] Evrall, N. (2004), Depth profiling with confocal Raman microscopy, Part II. Spectroscopy-Springfield then Eugene then Duluth, 19, 16-27

Oil PMMA PS PET

Air Tape Glue Silicone

a) Oil b) Air c) T ape d) Silicone Layer

36

SEM Characterization of functionalized Carbon Nanotubes

Philipp Siedlaczek, Gerald Singer, Gerhard Sinn, Leon Ploszczanski, Helga Lichtenegger

University of Natural Resources and Life Sciences Vienna, Institute of Physics and Material Sciences, Peter-

Jordan-Straße 82, 1190 Vienna, Austria

Carbon nanotubes are high performance materials which exhibit superior mechanical, electrical and

thermal properties. Due to their outstanding metallic and semi-conducting behaviour, research on a

large scale is currently done for electronic applications. Semiconductors, batteries, capacitors and

photovoltaic systems are just a few future prospects of their field of application. However, attention

is also paid to their mechanical properties since the measured tensile strength can achieve up to

63GPa, which is multiple times more than steel. This quality can be used to reinforce carbon fibre

composites substantially.

In order to utilize carbon nanotubes adequately in composites, the nanotubes have to be modified.

Their strong nonpolar character refuses any interaction with epoxy resins, the important polymer for

adhering carbon fibre mats. Thus, chemical treatment of nanotubes is necessary to attach functional

groups, such as carboxylic groups and/or amine groups, at the surface of the molecule via oxidation to

induce intermolecular linkages to epoxy groups or amine groups of the hardener. After the reaction,

the mechanical properties along with thermal and electrical conductivity can be exploited to a great

extent. Common oxidation methods contain aggressive chemicals such as sulfuric acid, nitric acid,

potassium permanganate and sodium nitrate in large quantities. The exhaust gases and side products

are hazardous and toxic. Too harsh treatments additionally damage the carbon nanotubes and

subsequently reduce its mechanical properties tremendously.

This master thesis follows a gentler, more economic and environmental friendlier approach with H2O2

as an oxidizing agent. Analysis with Scanning Electron Microscopy (SEM) and Energy Dispersive X-Ray

Spectroscopy (EDX) offer a powerful method to determine successful oxidation techniques.

Furthermore, it is possible to analyse the degree of destruction and the expected ability to form

crosslinks with epoxy resins.

37

FEBID Based Direct-Write of 3D Plasmonic Gold Structures

Robert Winkler(1), Franz Schmidt(1,2), Ulrich Haselmann(1), Jason Fowlkes(3,4), Philip Rack(3,4), Harald

Plank(1,5)

(1) Graz Centre for Electron Microscopy, Steyrergasse 17, 8010 Graz (2) Institute of Physics, Karl-Franzens-University, Universitätsplatz 5, 8010 Graz

(3) Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA (4) Department of Materials Science and Engineering, University of Tennessee, Knoxville, Tennessee, USA

(5) Institute for Electron Microscopy, Graz University of Technology, Steyrergasse 17, 8010 Graz

During the last decade, resonant optics attracted enormous interest in science and technology as this

research field provides deep insights in fundamental physics but also an increasing number of

applications ranging from filters over waveguides towards sensor devices. While several techniques

for the fabrication of metallic structures have been introduced [1] the direct-write fabrication of highly

defined structures on the nanoscale, especially for complex three-dimensional geometries on non-flat

surfaces is still an intractable challenge. Focused Electron Beam Induced Deposition (FEBID) recently

has taken a huge step forward in terms of fabrication of predictable and complex three-dimensional

geometries, leveraging this technique into the status of a nano-printer [2,3] not only for 2D but also in

real 3D on almost any substrate material and morphology. Beside the reliable shape performance on

the nanoscale high purity of the material is essential for plasmonic activity. Therefore, the direct usage

of FEBID structures for plasmonic investigations is impossible due to the high carbon impurities of

about 90 at.% [4].

In this contribution we focus on the fabrication of complex freestanding FEBID structures (Fig. 1) in

general and in particular for the plasmonic investigation. In this context an alternating point sequence

approach is presented that enables 3D-nanoprinting beyond current limitations. In the following we

briefly show a purification process to Au precursor Me2Au(acac) as an ideal material for plasmonics

leading to pure Au structures in general. Applying the described purification process with adapted

parameter to such 3D deposits leads to compact and pure Au structures. In the last step, we present

TEM based EELS measurements to show plasmonic activity. This demonstrates the potential of FEBID

as fabrication method for free-standing, three-dimensional plasmonic structures on practically any

given surface. By that, the field of resonant optics can be expanded by yet unknown 3D architectures

in combination with regions which were very complicated to access in the past.

Figure 1: 3D-nanoprinting of plasmonic active FEBID-structures: First, complex 3D-nanoarchitectures

are reliably fabricated (left). After that a purification step utilizing electron stimulated reactions with

water vapor is introduced to transfer the Au-C deposition into pure gold as shown via TEM

characterization (center). Finally, STEM-EELS investigation revealed plasmonic activity (right).

[1] L. Hirt, A. Reiser, R. Spolenak, and T. Zambelli, (2017) Adv. Mater. 1604211. [2] J. D. Fowlkes, R. Winkler, B. Lewis, M. Stanford, H. Plank, P. D. Rack (2016) ACS Nano, 10, 6163-6172. [3] R. Winkler, F. P. Schmidt, U. Haselmann, J. D. Fo