bluuebunny/crossref_metadata_2025 huggingface dataset:
In‐situ propagation of a
Cu
phase in germanium nanowires observed by transmission electron microscopy
Authors: Hajraoui Khalil El , Zeiner Clemens , Robin Eric , Kodjikian Stéphanie , Lugstein Alois , Rouvière Jean‐Luc , Hertog Martien Den
Published: December 2016

Semiconductor nanowires (NWs) are promising candidates for many device applications ranging from electronics and optoelectronics to energy conversion and spintronics. However, typical NW devices are fabricated using electron beam lithography and therefore source, drain and channel length still depend on the spatial resolution of the lithography. In this work we show fabrication of NW devices in a transmission electron microscope (TEM) where we can obtain atomic resolution on the channel length using in‐situ propagation of a metallic phase in the semiconducting NW. The corresponding channel length is independent on the lithography resolution. We show results on semiconducting NW devices fabricated on two different electron transparent Si
3
N
4
membranes: a calibrated heater chip from DENs solution [1] and homemade membranes where the NW‐metal contact is locally heated by Joule heating [2]. We demonstrate a real‐time observation of the metal diffusion in the semiconducting NW. First we present results on in‐situ propagation of a copper‐germanium phase in Ge NWs while monitoring the system temperature [3] and by Joule heating while measuring the current through the device. We study the kinetics and rate limiting step by monitoring the position of the reaction front as a function of time. Second we will show characterization of the formed phase at atomic length scales with different (S)TEM techniques (electron diffraction tomography, energy dispersive X‐ray spectroscopy, HR(S)TEM) to understand how the metal atoms diffuse and incorporate into the germanide phase at the reaction front and how these parameters relate to the electrical properties of the same interface. Using EDX and diffraction characterization we find that an orthorombic Cu
3
Ge phase is created in the reacted NW part, see Fig. 2,3. Furthermore, both Cu and Ge are diffusing in opposite directions. Both EDX and diffusion studies indicate that the reaction proceeds via surface diffusion along the Cu
3
Ge segment.
Chemical arrangement and surface effects in
CoAu
nanoparticles
Authors: Tournus Florent , Loiselet Ophelliam , Sato Kazuhisa , March Katia , Stephan Odile
Published: December 2016

Nanoparticles associating a noble metal and a ferromagnetic metal are appealing from a magneto‐plasmonics point of view, in addition to the problematics of magnetic anisotropy tailoring (interface anisotropy, phase transformation) and of nanoalloy original geometries. Because Co and Au are immiscible in the bulk phase, and since fcc cobalt and gold have highly different cell parameters, chemically separated structures (core‐shell type) are expected for nanoparticles.

We have studied CoAu cluster assemblies, with a diameter between 3 and 10 nm, prepared by low energy cluster beam deposition (LECBD) where nanoparticles are formed in out‐of‐equilibrium conditions by laser vaporization, then deposited on a substrate under ultrahigh vacuum conditions and protected by a capping layer (amorphous carbon, to avoid oxidation). The nanoparticles' structure and chemical arrangement (see figures) have been investigated by HRTEM, STEM‐HAADF and STEM‐EELS before and after annealing (2h around 500°C). A mapping of the low energy electronic excitations has also been performed by STEM‐EELS, which is a challenge on such small nano‐objects.

As prepared particles are found to be inhomogeneous (as deduced from EELS measurements), with interatomic distances always corresponding to pure gold, and they appear to be surrounded by a shell of lighter HAADF intensity which rapidly transforms upon electron beam exposure in STEM. After annealing, a phase separation is observed and CoAu nanoparticles adopt a core‐shell structure where, as observed by HRTEM (with a clearly visible difference of inter‐plane distances between Co and Au regions), STEM‐HAADF and STEM‐EELS, an off‐centered cobalt core is surrounded by a gold shell (see figure). For both as‐prepared and annealed nanoparticles, the Co is not oxidized thanks to the efficient protection of the thin carbon layer. Using low‐loss STEM‐EELS mapping, we are able to observe some surface contributions, which may reflect collective electronic resonances (surface plasmons), with significant differences between as‐prepared and annealed CoAu nanoparticles (in particular, an intense peak is detected on the gold‐rich regions, around 6 eV).

These results shed light on the atomic‐scale behavior of the Co‐Au nanoalloy, here in an amorphous carbon matrix, which will be compared to other dielectric matrices, and will help us to understand the original magnetic properties of these hybrid nanoparticles.
Deconvolution of
EDS
steel spectra using low acceleration voltages and low energy
X
‐ray lines
Authors: Terborg Ralf , Salge Tobias , Pinard Philippe , Richter Silvia
Published: December 2016

One of the most important materials used in industry is steel. Its fine microstructure consisting of different phases and inclusions, has led to the development of new steel alloys that push the analytical requirements for spatial resolutions down to the 100 nm scale. In X‐ray microanalysis either using energy or wavelength dispersive spectrometry (EDS, WDS), low accelerating voltages, e.g. ≤ 6 kV, can fulfill this requirement. At low accelerating voltages only the L lines for most steel alloying elements (e.g. Cr, Mn, Fe, Co, Ni) can be determined as their K lines can no longer be excited. Qualitative and quantitative analysis of low energy L lines is very challenging due to the presence of absorption edges within the bremsstrahlung background, the energy dependence of the efficiency and the uncertainties of absorption effects [1]. Peak overlaps, furthermore are considerable for EDS and can even be significant for WDS.

Due to these effects this type of analysis poses difficulties for EDS and WDS. To evaluate and compare these challenges for both techniques, a systematic study was performed on steels at acceleration voltages of 15 kV and 5‐6 kV [1,2]. Spectra and net intensities of 15 steel samples covering a wide range of concentrations of the major elements were simultaneously acquired on a JEOL JXA8530F electron microprobe equipped with 5 WD spectrometers and a Bruker XFlash ED spectrometer.

The results of the 15 kV spectra show that for elements with a concentration of < 1 wt.% the WDS results are more accurate when significant peak overlaps occur in ED spectra (Mn, Co, Cu). For elements without peak overlaps, EDS results were comparable (Si, Mo) to WDS [2]. At an acceleration voltage of 6 kV, the deconvolution results of ED spectra show agreement with the experimental spectra, fig. 1. Since the quantification of L lines is more sensitive to the quantification procedure and fundamental parameters (e.g. mass absorption coefficient) used, the comparison focuses on k‐ratios instead of the quantified mass fractions. Fig. 2 shows a good agreement between EDS and WDS k‐ratios, substantiating the peak deconvolution procedure shown in fig. 1.
Platinum shadowing for correlative light and electron microscopy
Authors: Mateos Jose Maria , Guhl Bruno , Doehner Jana , Barmettler Gery , Kaech Andres , Ziegler Urs
Published: December 2016

Fluorescence microscopy reveals molecular expression at nanometer resolution but lacks ultrastructural context information. Electron microscopy provides this contextual subcellular details but protein identification requires elaborate protocols. Correlative light and electron microscopy produces complimentary information that expands our knowledge of protein expression in cells and tissue. Even though a number of correlative approaches are currently available, few of these allow subcellular localization in tissue because of the challenges with sample preparation and 3D complexity. Tokuyasu cryo‐sections (Tokuyasu, 1980) preserve the sample ultrastructure and antigenicity of most epitopes, however, heavy metal exposure generates weak contrast to the samples rendering often interpretation of the data difficult.

We present a quick, simple and reproducible method for protein localization by conventional and super‐resolution light microscopy combined with platinum shadowing and scanning electron microscopy to obtain topographic contrast from the surface of ultrathin cryo‐sections collected on silicon wafers. Figure 1 shows protein distribution at nuclear pores in the topographical landscape of mouse kidney tissue.
New approach for low dose electron diffraction tomography
Authors: Tietz Hans , Sparlinek Peter , Olyenikov Peter , Ghadimi Reza
Published: December 2016

Due to strong interaction of electrons with matter, 3D electron diffraction tomography has been proven to be a reliable method to solve structures of very small crystals, compared to X‐ray diffraction; even for small protein crystals [1].

Several procedures for automated acquisition of tomographic diffraction data sets have been described [2, 3], using precession technique or discrete beam tilt perpendicular to the goniometer tilt axis. However, those mentioned methods are mainly used for less beam sensitive materials and are not suitable for low dose applications.

Shi et al. [4–7] refined the collection of 3D data sets under low dose condition (less than 10 e
−
/Å
2
). Here, the camera system is continuously acquiring diffraction data during a continuous tilt of the goniometer. This allows to completely scan the Fourier space. It turned out that the stability of the goniometer tilt speed and the limited flexibility of the tilt acquisition parameters are problematic.
Structural special features of the gold nanolayer sputtered on the glass surface modified by surface ion exchange and chemical etching
Authors: Sidelnikova Olga , Salanov Aleksei , Yatsenko Dmitriy , Serkova Alexandra
Published: December 2016

The research data are related to X‐ray structure analysis of the gold nanolayers (50, 100 and 200 nm thick) on glass surface modified by the surface ion exchange, chemical etching or by treatment combining two indicated above methods. The soda lime silica glass has been modified by the special Surface Ion Exchange Paste (SIEP) [1‐3]. Na
+
/Li
+
ion exchange with SIEP has included next stages: glass surface degreasing and washing; the SIEP laying on the glass surface; thermal treatment at ~ 300º C for 15 – 20 min.; washing with running and distilled water. The chemical etching of glass have been carried out with the composition containing hydrofluoric acid [4]. The glass surface morphology has been observed by the SEM JSM‐6460 (Jeol, Japan); the gold nanolayers were deposed on glass surface by the special device JVC‐1600 (Jeol, Japan).

X‐ray data have been measured with diffractometer D8 Advance (CuKa radiation, one‐dimensional detector Lynx–Eye with nickel filter). The range of the measurement was 2q = 10 – 120° with step 0,02° and acquisition interval 35,4 c. The program Topas 4.2 (Bruker AXS, Germany) and initial structural data of inorganic base ICSD, FIZ Karlsruhe, Germany have been also used in calculations.

X‐ray data analysis allows make conclusion that the surface modification of the soda lime silica glass by the surface ion exchange and chemical etching has a substantial influence upon the size of gold crystal grains (Figure 1 i). Gold crystal grains have elongated shape in direction . The increase of the gold nanolayer thickness leads to decreasing of the crystal grains size. The least size have been related to gold nanolayer 200 nm thick on glass surface modified by combined method indicated above: in direction average calculated grains size is ~ 14.7 nm; grains size averaged in directions , and is ~ 6.8 HM.

Authors express thanks to N.V. Bulina for technical support.
Cobalt‐cerium coating formation: from
ESEM
to
TEM
analyses
Authors: Poitel Stéphane , Zhu Jun , Hébert Cécile , Herle Jan Van , Willinger Marc
Published: December 2016

Fuel cells are promising devices for clean energy conversion. In particular, Solid Oxide Fuel Cells (SOFC) convert natural gas and biogas into electricity and cogenerated heat with high efficiency. Their working temperature (700–800°C) may induce performance degradation in the cells and other components of the stack (series connection of cells). Chromium contamination of the cathode (oxygen electrode) is one of these degradation processes in SOFC. This chromium evaporates from the steel bipolar plates which act as cell‐to‐cell interconnections. To suppress the Cr evaporation, the steel plates must be coated. Understanding the degradation phenomena in the stack and their evolution helps to improve the device lifetime. Post test analyses are not always sufficient but already give good insight. On the other hand, the possibility to reach operating temperature (up to 900°C) and to mimic operating conditions of oxidising and reducing atmospheres (up to 30 Pa of O
2
or H
2
) in environmental SEM allow to observe the evolution in near real condition. In this study, Sandvik Material Technology's cerium‐cobalt protective PVD coating on top of the Sandvik SSHT steel (FeCr‐base) is analysed. Several movies were recorded during Cr‐oxidation growth underneath the coating for 48 hours in an ESEM in oxidising conditions (900°C, 30Pa O
2
). Growth observation of the oxide layer allows a better understanding of the behaviour of the steel with this protective layer. Pore stability, elemental diffusion through layers and the probable formation of new phases were observed by SEM and EDX. FIB was used for cross section observations and will be used for TEM lamella preparation in future. TEM observation will allow to analyse the formed crystals by diffraction and establish a correlation with the observations from the ESEM experiments.
The supramolecular packing of the gel‐forming
MUC5B
and
MUC
2 mucins and its importance for cystic fibrosis
Authors: Nilsson Harriet E. , Trillo Sergio Muyo , Ermund Anna , Bäckström Malin , Thomsson Elisabeth , Ambort Daniel , Koeck Philip J. B. , Thornton David J. , Hansson Gunnar C. , Hebert Hans
Published: December 2016

Text:

Introduction:
The genetically related gel forming mucins, MUC2 (intestine), MUC5B (airways), MUC5AC (airways, stomach) and MUC6 (stomach) have large sizes with heavily glycosylated mucin domains in the central part. The C‐termini form intermolecular dimers. The N‐terminal regions are evolutionarily similar with identical domain organization important for the oligomerization. MUC5B is vital for normal mucociliary clearance of the lungs whereas MUC2 in colon forms an inner dense and attached stratified layer impermeable to bacteria, and an outer loose and unattached layer habituating commensal bacteria. The MUC2 N‐terminus (D1‐D2‐D′D3 domains) was shown to form concatenated polygone‐structures under low pH‐ and high calcium conditions (1).

The Aim is to understand the cellular packing of the MUC5B and MUC2 mucins and how this influences their secretion.

Method:
The N‐terminal and D′D3 domains of MUC2 and MUC5B were expressed, purified and then analyzed by subsequent gel filtration, transmission electron microscopy and single particle image processing.

Results:
MUC5B multimerizes by disulfide bonds between the D3‐domains giving the MUC5B polymer a linear structure (2). Analysis of the MUC5B N‐terminus at lower pH and higher calcium concentration revealed a tight dimer+dimer packing where the second dimer is turned upside down by 180 degrees and then slightly rotated. This way of packing the MUC5B in the granulae will allow a slow unwinding of a linear molecule.

Conclusion:
The MUC5B and MUC2 mucins are packed in the mucin granulae in a way allowing the formation of linear strands or net‐like structures, respectively.
Power ofFIB‐SEMTomography for Biological Samples
Authors: Kizilyaprak Caroline , Niedergang Florence , Bellis Damien De , Blanchard Willy , Daraspe Jean , Geldner Niko , Humbel Bruno
Published: December 2016

Three‐dimensional (3‐D) spatial distribution of organelles within cells at nanometer resolution is essential to better understand cellular processes and functions. Currently, FIB‐SEM tomography provides a promising technology to generate volume data with nanometer resolution (Bushby et al., 2011; Heymann et al., 2009; Knott et al., 2011; Villinger et al., 2012; Wei et al., 2012). A FIB‐SEM microscope is a scanning electron microscope (SEM) combined with a focused ion beam (FIB) where both beams coincide at their focal points. This combination enables to locally section bulk embedded‐resin samples by ion milling, creating a fresh surface for subsequent imaging with the electron beam. This process is repeated automatically to generate 3‐D information of relatively large volumes with a field of view of several micrometers.

Any stained and embedded resin samples prepared for transmission microscope can be used for FIB‐SEM tomography offering the possibility to visualise directly in 3D a wide range of biological samples and typically volumes with a pixel size of 10 nm can be achieve (Figure1). However, to obtain a better resolution, considerations have to be given concerning ultrastructure preservation and samples preparation (Kizilyaprak et al., 2015). High pressure freezing (HPF) combined with freeze‐substitution (FS) and resin embedding constitutes a method of choice to find the best compromise between ultrastructural preservation and high contrast of cellular components (Figure2).

In conclusion, we propose biological sample preparation protocols that can serve as starting point to visualize in 3‐D wide range of biological samples at nanometer resolution including HPF/FS samples and correlative microscopy approach using FIB‐SEM Tomography.
Towards imaging of defects in diamond by high‐resolution
TEM
Authors: Leiter Robert , Qi Haoyuan , Biskupek Johannes , Naydenov Boris , Jelezko Fedor , Kaiser Ute
Published: December 2016

Nitrogen‐Vacancy (NV), silicon‐vacancy (SiV), germanium‐vacancy (GeV) and other colour centres in diamond have been of rising interest in recent years due to their potential applications such as quantum information processing [1] and fluorescent labelling in biology. For such applications, it is necessary to incorporate the colour centres in diamond thin films or nanodiamond particles in a controllable way. Since the colour centres are formed by single atomic vacancy and single substitution, one fundamental step towards controlled incorporation is to characterize the structure of the colour centres with high spatial resolution. Although structural characterisation of NV‐centres has been accomplished by optical and magnetic resonance methods, atomic structures of other colour centres often remain elusive.

Aberration‐corrected high‐resolution transmission electron microscopy (AC‐HRTEM) has proven to be a powerful technique for structural analysis with atomic resolution. However, the imaging conditions necessary to directly image the structure of an SiV, GeV or other colour centres remain unclear. Here we explore the optimum imaging conditions for the detection of SiV and GeV centres by using HRTEM image simulation. In order to maximize the image contrast from the SiV and GeV centres, we carried out a series of HRTEM simulations under various imaging conditions such as crystal orientation, acceleration voltage, spherical aberration coefficient (C
s
) and defocus. The HRTEM image simulation was implemented using a multislice algorithm in the QSTEM software package [4].

Our results show that it should be possible to directly image both SiV and GeV centres in [120] projection at an acceleration voltage of 300 kV (see Figures 1 and 2, respectively). Under negative C
s
imaging conditions (C
s
= ‐15 µm and slight overfocus), both SiV and GeV centres can be clearly detected. Due to the higher atomic number of Germanium, GeV centres provide slightly higher contrast as compared to SiV centres.
OptimizingCLEMprotocols for plants cells: A good preservation ofGFPfluorescence and ultrastructure in Arabidopsis roots
Authors: Marion Jessica , Bars Romain Le , Satiat‐Jeunemaitre Béatrice , Boulogne Claire
Published: December 2016

While there is an explosion in Correlative Electron and Light Microscopy (CLEM) protocols, many have been developed on different eukaryotic models, though reports on plant cell exploration by CLEM remain scarce (Bell et al., 2013). Indeed, plant cells represent a challenge for imaging in many ways. Plant specificities have to be taken into account when optimizing CLEM protocols. To name some, strong autofluorescence of cellular components specifically present in plant cells (chlorophyll, pigments, etc.) and interfering with fluorescent signals of reporter proteins, presence of a large central vacuole complicating the fixation and the dehydration processes needed for TEM processing, cell wall and cuticles acting as physical barriers and impairing resin embedding processes (Hawes et al., 2001).

Here we report two “in hand” robust and easily reproducible protocols to make such CLEM approaches on plant material. The proposed protocols had been worked out on the delicate question of autophagosomes identity in plant cells. Autophagy is a degradation process of intracellular components mainly implicated in response to environmental stresses. It consists in the delivery of cytosolic cargos entrapped by a compartment, named autophagosome, to lytic compartments for degradation and recycling. In plant cells as in any eukaryotic cells, autophagosomes have been identified as ring‐shape or punctate structures, thanks to the light imaging of reporter proteins of markers for autophagosomes (Le Bars et al., 2014).

This study is using a Arabidopsis transgenic lines stably expressing GFP‐ATG8, which is a common marker for autophagosomes. Two CLEM protocols are used: firstly the high pressure freezing followed by acrylic resin embedding, and on the other hand the Tokuyasu method. Here we compare the performance of the two protocol to preserve GFP fluorescence and enhanced ultrastructural features in plant cells.
High Resolution Chemical Imaging on the Helium Ion Microscope
Authors: Dowsett David , Audinot Jean‐Nicolas , Vollnhals Florian , Eswara Santhana , Wirtz Tom
Published: December 2016

The ORION NanoFab helium ion microscope (HIM) has become an ideal tool for imaging and nano‐patterning tool owing to its high lateral resolution [1]. Helium ions lead to resolutions of 0.5 nm for SE based imaging, while structures with sub 20 nm feature sizes may be rapidly patterned using Ne. Despite these advantages, the analysis capability of the machine is currently limited. At beam energies of 35 kV helium ions do not lead to the emission of characteristic X‐rays from a sample. While some compositional information can be obtained from back scattered helium [2], identifying elemental information is more difficult. Secondary Ion Mass Spectrometry (SIMS) is a powerful ion beam based technique for analyzing surfaces with high sensitivity and high mass resolution. SIMS is based on the generation and identification of characteristic secondary ions by irradiation with a primary ion beam (in this case helium or neon). The typical interaction volume for SIMS is around 10 nm in the lateral direction. As the probe size in the HIM is substantially smaller (both for He and Ne) the lateral resolution is limited only by fundamental considerations and not, as is currently the case on commercial SIMS instruments, the probe size [3‐4]. The prospect of adding SIMS to the HIM yields not just a powerful analytical capability, but opens the way for in‐situ correlative imaging combining high resolution SE images with elemental maps from SIMS [5].

We have developed a prototype SIMS spectrometer specifically adapted to the HIM. Notably the instrument is capable of producing elemental SIMS maps with lateral resolution limited only by the fundamental interaction between the primary beam and the sample. All elements/isotopes and small clusters with masses up to 500 amu are detectable with a mass resolution M/ΔM greater than 400 and parallel detection of 4 mass channels.

In this presentation we will introduce the HIM‐SIMS technique and present the latest application results in the field of materials science.
ZnO
Nanostructures for Mid‐
IR
Plasmonics
Authors: Taha Mohamed Hamza , Bluet Jean , Masenelli‐Varlot Karine , Langlois Cyril , Epicier Thierry , Bugnet Matthieu , Canut Bruno , Boisron Olivier , Melinon Patrice. , Masenelli Bruno
Published: December 2016

Degenerate metal oxide nanocrystals (NCs) are promising systems to expand the significant achievements of plasmonics into the infrared (IR) range
1
. We report on the tunable mid IR Plasmon induced in degenerate Al and Ga doped ZnO (AZO and GZO) nanocrystals. The NCs were obtained by Low Energy Cluster Beam Deposition (LECBD). By varying the Al and Ga content from 3 to 9 at.% within the particles we are able to tune the plasmon wavelength from 3 to 4 μm. However, the plasmon resonances are characterized by an unusually large damping, which originates from two mechanisms. The first one is the Oriented Attachment (OA) process (cf. figure 1, B)
3
. When NCs attach by epitaxy, the resulting structure has a lower symmetry, which induces a shift and broadening of the plasmon resonance. Embedding the particles in an Al
2
O
3
matrix has prevented the OA, and hence the damping was reduced along with broadening. The second mechanism is the partial activation of the dopants. We have observed that less than half of the dopants actually participate to the electron gas
2
. The cause of the partial activation is related to the position of the dopants within the particles. It has been proposed that the damping is larger if the dopants are homogeneously distributed
4
. In the present work we investigate the possibility of mapping the spatial distribution of dopants within the nanocrystals usingFEI‐TITAN ETEM equipped with High resolution Gatan GIF (see Figure 2). We subsequently anneal the nanocrystals to let them reach the thermodynamic equilibrium. The distribution of dopants, and its consequences on the plasmon resonances is then investigated [5,6].
In vivo structural characterization of bacteriophage 812K1‐420 virion lytic cycle intermediates
Authors: Peterek Miroslav , Novacek Jiri , Plevka Pavel
Published: December 2016

Staphylococcus aureus is one of the most important bacterial pathogens causing a wide spectrum of diseases varying in severity. Treatment of the infections is often complicated by S. aureus resistant to antibiotics. Current development of antibiotics is not fast enough to counterpart the emergence of antibiotic‐resistance. Preclinical studies showed, that phage therapy is promising alternative for treatment of bacterial infections including those caused by resistant strains, but approval of bacteriophage‐based drugs requires a detailed functional understanding of the molecular mechanisms of phage infection which we are still lacking.

Bacteriophage 812K1‐420 from the family Myoviridae is potential candidate to be used in phage therapy against S. aureus. Polyvalent and strictly virulent bacteriophage 812K1‐420 virion is composed of an icosahedral head containing linear 145,5 kb dsDNA, contractile tail and baseplate which specifically recognizes specific surface on the bacterial host. Replication and virion‐assembly mechanisms inside host cell are not fully explored yet, although few bacteriophage 812K1‐420 lytic cycle intermediates of virion assembly were reproduced in vitro.

Here we present initial structural analysis of bacteriophage 812K1‐420 infection in vivo using cryo‐electron tomography. We used cryo FIB milling technique to thin plunge frozen S. aureus cells (600‐1000 nm in diameter) infected by phage 812K1‐420 into wedge geometry . The cryoET data acquired on FEI Titan Krios microscope and tomograms were reconstructed using program IMOD.
Full structural and chemical characterization of the uniaxial relaxor
SBN
‐67 (Sr0.67Ba0.33Nb2O6)
Authors: López‐Conesa Lluís , Rebled José Manuel , Ruiz‐Caridad Alicia , Dezanneau Guilhem , Torres‐Pardo Almudena , Ruiz‐González Luisa , González‐Calbet José Maria , Estradé Sonia , Peiró Francesca
Published: December 2016

SBN belongs to the tetragonal tungsten bronze (TTB) family of uniaxial ferroelectric relaxor materials, with a single component polarization vector pointing along the tetragonal
c
axis. The high dielectric constants observed and polarization controlling around the ordering temperature 'T
m
' make of relaxors promising systems for applications such as electrocaloric, pyroelectric, piezoelectric/electrostrictive devices and sensors.

The idealized structure of SBN, with the generalized formula A
2
B
4
C
4
Nb
2
Nb
8
O
30
, has three types of structural channels along the
c
axis, formed by two types of crystallographically independent NbO
6
octahedra. Disorder due to presence of voids on A cation sites (occupied by Sr atoms) and random distribution of Ba and Sr on B cation sites are thought to be responsible for the relaxor behavior, via the formation of random electric fields and, subsequently, polar nanodomains oriented in the only easy polarization axis
c
, without a structural change occurring in the phase transition. In this work we present structural and chemical studies of a SBN single crystal by means of Selected Area Electron Diffraction (SAED), High Angular Annular Dark Field (HAADF), Annular Bright Field (ABF) and Electron Energy Loss Spectroscopy (EELS) along the [001] and [100] zone axes. Experiments were carried out in non‐aberration corrected (Phillips CM30, JEOL J2100, JEOL2010F) and probe‐corrected (Titan
3
G2, ARM cF200) TEMs.

An incommensurate structural modulation in SAED experiments along the [100] direction already points to structural disorder (Fig. 1). Diffuse scattering in the form of streaking patterns in the main reflections can also be related to strain induced by the presence of vacant sites. HAADF imaging along the [100] zone axis directly shows in real space the presence of cationic vacancies in A‐type sites. Elongation of the atomic columns in the center of the B pentagonal sites in HAADF images along the [001] zone axis (Fig. 2) is compatible with the presence of Ba and Sr randomly occupying that crystallographic position. EELS spectrum imaging along the [001] zone axis (Fig. 3) directly confirmed the mixing of Sr/Ba in the B‐type sites. With these results, the causes for local charge disorder in the structure are mapped in direct space with atomic column resolution. ABF imaging allowed mapping the oxygen sublattice in the structure, achieving the full structural characterization of the material.
How the Detector Geometry Influences
EDXS
Quantification
Authors: Kraxner Johanna , Schäfer Margit , Röschel Otto , Paller Manuel , Haberfehlner Georg , Kothleitner Gerald , Grogger Werner
Published: December 2016

Thanks to the recent advent of very large area sensors and multi‐sensor systems X‐ray spectrometry in analytical electron microscopes received a substantial boost. In particular, the combination of 2 or 4 detectors has proven to obtain such a good sensitivity that the detection of single atoms is feasible [1]. However, if it comes to quantitative work partial or full shadowing of one or more detectors by the specimen holder will introduce systematic errors of the obtained results [2].

In this paper we closely investigate the influence of the detector‐microscope geometry on quantification using a ChemiSTEM system (Super‐X) on a Titan microscope with a low background double tilt high visibility holder [3]. We introduce a procedure, which allows for the first time to determine experimentally the detector positions inside the microscope chamber in terms of elevation angle, azimuthal angle, and distance. We measured the amount of shadowing as a function of specimen tilt angle α and compared these measurements to simulations, where we varied the detector positions in order to optimize the fit to the experimental data. The sample was a specially designed Au‐Pd thin film deposited on a silicon nitride membrane grid. The obtained positions were then verified by comparing the results to data obtained from different positions of the specimen holder.

To measure the amount of shadowing we used normalized intensities of the available X‐ray lines. An optimized simulation for one of the Super‐X detectors (Q1) may be seen in fig. 1 (Si‐K data). By comparing measurements from different lines we noted a significant difference depending on the X‐ray energies (compare Si‐K and Au‐L data in fig. 1). By further investigating this issue we found that the Beryllium specimen carrier inside the specimen holder may turn transparent for high enough X‐ray energies (e.g. Au‐L). In other words, the amount of shadowing not only depends on tilt angles and specimen positions but also on the energy of the X‐ray line of interest (see fig. 2).

Once the detector positions are known the shadowing effects can be taken into account and their influence on quantitative results can be corrected. In this paper we characterize potential effects and systematic errors they introduce, which are relevant to conventional EDXS work as well as analytical tomography experiments. The procedures described will transform EDXS on a multi‐detector system from qualitative analysis to full quantitative analysis.
Structural characterization of the human potassium channel Kir 2.1
Authors: Dezi Manuela , Wagner Renaud , Bendahhou Said , Venien‐Bryan Catherine
Published: December 2016

Inwardly‐rectifying potassium (Kir) channels regulate membrane electrical excitability and K+ transport in many cell types where they control such diverse processes as heart rate, vascular tone, insulin secretion and salt/fluid balance. The physiological importance of eukaryotic Kir channels is highlighted by the fact that genetically‐inherited defects in Kir channels are responsible for a number of human diseases such as in Andersen's syndrome (Kir2.1), Bartter syndrome (Kir1.1), and neonatal diabetes (Kir6.2). To date, the available treatment is unfortunately not rational but rather empirical and this is mostly due to the lack of knowledge about atomic structure of these channels.

To elucidate how channel function becomes defective in the disease state requires a detailed understanding of channel structure in both the open and closed states. We have reported the structure of a homologous bacterian KirBac3.1 potassium channel with an open bundle crossing indicating a mechanism of channel gating determined by X‐ray crystallography at 3Å resolution. In this model, the rotational twist of the cytoplasmic domain is coupled to opening of the bundle‐crossing gate via a network of inter‐ and intra‐subunit interactions [1,2]. In addition, we have also used EM analysis of 2D crystals of the same Kirbac channel trapped in an open state and compared these results with the 3D structure [3].

We are now focusing in characterizing the structural determinants correlated to malfunctioning behind Andersen mutants forms. We are therefore studying the structure of the human potassium channel kir2.1.The full‐length human Kir2.1 (50 kDa the monomer, 200kDa the tetrameric functional form) was over expressed in yeast
Pichia Pastoris
and subjected to various method for its characterization.

Here we show preliminary results on the expression, the purification and the imaging of negatively stained isolated kir 2.1 in detergent. The particles have an averaged size of 12 nm consistent with a tetrameric form of the native channel. The oligomeric organisation is supported by native gel electrophoresis and Dynamic Light Scattering measurements.

Our results will hopefully contribute to uncover the mechanism of clinically‐relevant disease‐causing mutations on the structure, dynamics, and function of kir2.1 potassium channels and at investigating the potential of pharmaceutical correctors.
High‐resolution identification of immuno‐labelling nanoparticles on tissue using
X
‐ray detection
Authors: Scotuzzi Marijke , Kuipers Jeroen , Wensveen Dasha I. , de Boer Pascal , Hagen Cornelis W. , Giepmans Ben N G , Hoogenboom Jacob P.
Published: December 2016

Cellular structure can be imaged at nanometer resolution using electron microscopy, but the biological molecules remain invisible in grayscale electron micrographs. With Correlative Light and Electron Microscopy (CLEM)[1], fluorescence recorded on a separate light microscope can be used to identify molecules in colour, but the resolution gap with electron microscope precludes accurate localization. The use of super‐resolution fluorescence microscopy techniques combined with CLEM[2,3] holds great promise but is still one order of magnitude off in resolution compared to EM. In addition, accurate registration of the separate images may be challenging and correlation may require expert procedures to maintain, e.g., protein photo‐switching under EM preparation conditions. Thus, while CLEM adds information represented in the colors of visible light, molecular identification and localization at the resolution of the electron microscope is still hampered. Therefore, we are exploring novel approaches to achieve color‐EM using the electron beam to add information about the specimen to the electron micrographs.

Detection of electron‐beam excited luminescence has been explored in the past. Cross‐sections for visible luminescence (also called cathodoluminescence) are, however, small and most biological fluorescent labels are destroyed before sufficient signal has been collected to allow high‐resolution localization [4]. Phosphorescent nanoparticles are considered as non‐bleaching alternative, but particle sizes are not yet well‐defined and bio‐functionalization has in most cases still to be pursued[5].

Here, we explore detection of X‐ray fluorescence to identify nanoparticle labels[6] at EM resolution in colour on tissue. We use Au and CdSe colloidal quantum dots, immuno‐targeted to guanine quadruplexes, resp. insulin, as molecular labels in large‐scale electron microscopy of pancreatic tissue. Energy dispersive detection of X‐ray emission (EDX) during electron irradiation allows discrimination of both nanoparticles based on their elemental (Au vs Cd) composition. We will present electron microscopy images overlaid with false colour elemental maps displaying single nanoparticles. Separate resolution tests have shown that individual Au particles down to 3 nm in size can be detected using EDX, highlighting the potential for coloured identification of materials in biological electron microscopy, revealing both compositional and ultrastructural information at nanometer‐scale resolution.
In‐situ thermal stability and reactivity monitoring of Au nanoparticles using
Cs
‐Corrected environmental
TEM
Authors: Chmielewski Adrian , Prunier Hélène , Nelayah Jaysen , Amara Hakim , Creuze Jérôme , Alloyeau Damien , Wang Guillaume , Ricolleau Christian
Published: December 2016

For the production of novel or more efficient catalysts, the link between nanoparticle (NP) structure and catalytic performances need to be understood. Such an understanding requires tools that allow the “observation” with single‐atom sensitivity of the surface of real catalysts in response to reaction conditions. Indeed, when nanocatalysts operate in gas environment and high temperature, they do not remain static but undergo dynamic atomic‐scale processes (surface restructuration, oxidation…) which directly influence catalytic properties. In this work, we have studied the thermal stability of Au‐Cu nanocatalysts under oxidative and reductive environments and/or at high temperature using environmental aberration corrected TEM (ETEM).

Au NPs with controlled sizes, compositions and morphology were synthesized by pulsed laser deposition. Environmental gas TEM of nanocatalysts at the atomic scale was undertaken in an aberration‐corrected JEM‐ARM200F TEM with
in situ
gas/temperature conditions achieved by using a MEMS‐based nanoreactor (Protochips Inc.) and equipped with a high performance Gatan OneView camera enabling the capture of both high quality images and high‐speed videos of
in situ
events (512 x 512 pixels at 300 fps).

Figure 1 compares TEM images of Au NPs (a) at room temperature in vacuum, (b) at 300°C under O
2
atmosphere and (c) at 300°C under H
2
atmosphere. At room temperature, in vacuum, NPs are stable but once a gas, O
2
and/or H
2
is injected, coalescence phenomena appear. The coalescence continues at 100°C and 200°C. An additional phenomenon appears at 200°C with O
2
that does not with H
2
: particle faceting. At 300°C, under O
2
, all particles are well facetted (truncated octahedron). When we cool down the sample with O
2
, the faceting disappears. Thus, particles size and morphology are clearly dependent on temperature and gas nature with an increase in temperature favoring particle coarsening and exposure to an O
2
atmosphere leading to more facetted Au NPs.

Figure2 shows TEM images of Au NPs at 300°C (a) which are all facetted at this temperature (truncated octahedron) and at constant temperature of 850 °C at different times (b,c,d). The NPs become more spherical at higher temperature and began to evaporate at 850°C. One can determine the surface energy of the evaporating particles by means of the Kelvin equation
2
. There is a size dependence of the surface energy of the NPs, indeed, the surface energy increases with the size. For a particle radius greater than 4nm, one cannot apply the Kelvin model. Thereby, in order to understand the underlying mechanism of the behavior of a nanoparticle in this size range, simulations using the tight binding model are in progress. Also, by alloying gold with copper, we are studying the phase reactivity of AuCu nanoparticles as well.
Gastric digestion of milk protein gels as assessed by time‐lapse Synchrotron
UV
‐microscopy
Authors: Thévenot Jonathan , Floury Juliane , Jamme Frédéric , Panouillé Maud , Lutton Evelyne , Boué François , Dupont Didier , Feunteun Steven Le
Published: December 2016

Gastric digestion is the result of physical disintegration, acidic hydrolysis and enzymatic reactions leading to the release of nutrients which are absorbed in the upper intestinal tract. Protein is one of the essential macro‐nutrient and can be eaten in a great variety of forms (solubilized, cross‐linked, in their native or denatured states). Controlling food protein gelation conditions result in the formation of particles with specific structural features. Several
in vivo
and
in vitro
studies have shown an influence of the macro‐ and microstructure on the kinetics of milk protein hydrolysis. Nevertheless, the mechanisms by which the structure of dairy gels can affect the digestion kinetics remain largely unknown.

The aim of the study was to assess the part play by HCl and gastric enzyme (i.e. pepsin) during gastric digestion using a dynamic and label‐free imaging technique on the DISCO beamline of Synchrotron SOLEIL to visualize
in situ
the milk protein gels breakdown kinetics. The DISCO beamline uses the deep ultraviolet range to probe the intrinsic UV tryptophan fluorescence without the need of specific external probes. Two milk gels with the same protein concentration but different microstructures were prepared either by rennet or acid coagulation of non‐fat milk. The disintegration of the different networks was monitored under digestion at body temperature in simulated gastric fluids and the effect of the acidic environment uncoupled from the enzyme effect. The evolution of particle area and mean fluorescence intensity has been determined, and used to estimate the kinetics of food particles breakdown.

The kinetics of acid gel
in vitro
digestion was significantly reduced compared to rennet gel. Our data indicate that rennet gel has a two‐step behavior during the acidification phase with a swelling followed by a contraction of the particle, not observed for acid gel. In addition, these microstructural modifications of rennet gel affect negatively the enzymatic breakdown kinetics of particles compared to acid gel.

This study leads to original methodological developments both from the point of view of the acquisition of data and their joint analysis. Getting
in situ
information about digestion kinetics, microstructural transformation and enzymatic reaction, allow further analysis of the digestion process.
Soft matter, and the many flavours of diffraction tomography
Authors: Stewart Andy
Published: December 2016

With the arrival in 2009 of diffraction tomography for crystal structure solution [1] of sub‐micron sized crystals, there has been a renaissance in the application of electron diffraction, with diffraction tomography providing a relatively easy path to quantitative analysis of crystal structures, compared to more conventional electron diffraction approaches. The collection of diffraction tomography data occurs by tilting the crystal around an arbitrary axis, which runs counter to the traditional notions of electron diffraction from low index orientated zones. Given the success of the method, it has produced a series of derivative techniques on this theme, of collecting tomographic diffraction from around an arbitrary axis. Each of these methods comes with its strengths and weaknesses.

An investigation into the relative merits of the various diffraction tomography techniques, ADT[1], RED[2], EDT[3], Rotation method[4], MicroED[5]. With an emphasis on the application of the techniques for soft matter and radiation sensitive materials, such as organic and protein crystals. Evaluation of the nuances in data collection strategies, hardware requirements, specimen requirements, and optional components, will all be considered, with details of how they affect the experimental setup, data acquisition, and processing.

We will discuss the need for validation of structural models and the added difficulties presented in doing so with soft matter materials, often leading to the requirement of validation by additional techniques.
An in situ multiscale approach for the characterization of plasticity and damage in a
TA6V
alloy
Authors: Cazottes Sophie , Dancette Sylvain , Bourlot Christophe Le , Maire Eric , Paris Thomas
Published: December 2016

Due to its specific strength and good biocompatibility, Ti‐6Al‐4V alloy find many applications in medical and aeronautic industries.

At room temperature, the studied TA6V alloy presents a bi‐modal microstructure consisting of hcp alpha phase together with bcc beta phase. When subjected to high cooling rates such as in welding processes, a lamellar hcp alpha' phase appears, which modifies the plastic and damage behavior of the alloy. In order to better understand the role of microstructure on failure of the material, a clear connection must be made between microstructural parameters such as interfaces, grain or lath orientation, grain boundaries, morphologies and failure initiation.

The deformation behavior of both welds and base metals were characterized by in situ EBSD tensile tests. Slip and twin systems were identified for bimodal and fully martensitic microstructure. Deformation paths were followed upon deformation, and related to original laths or grain orientation, see Figure 1. At higher deformation levels, damage starts and cavity appear equally at grain boundaries, phase boundaries, or inside grains. However, if a large number of information is available from these 2D observations, 3D characterization is required for a quantitative damage study as most of the cavities initiate in the volume and not at the surface. The same samples were then characterized by in situ tensile X Ray Tomography tests conducted at ESRF Grenoble.

For several deformation levels, a quantification of the cavity number and size was performed and a clear description of the cavity initation, growth, and coalescence was obtained in 3D, as reported on Figure 2.