bluuebunny/crossref_metadata_2025 huggingface dataset:
TEM
study of defect reduction in the growth of semipolar
GaN
grown on patterned substrates
Authors: Vennéguès Philippe , Tendille Florian , Khoury Michel , De Mierry Philippe , Mante Nicolas , Perez Jesus Zuniga , Feuillet Guy , Delaye Vincent , Martin Denis , Grandjean Nicolas
Published: December 2016

III‐nitride heteroepitaxial thin films and heterostructures suffer from the presence of large densities of structural defects, which are detrimental for the development of efficient devices. These defects result from differences between III‐nitride films and foreign substrates (structure, chemistry, lattice parameters). Transmission electron microscopy (TEM) is the technique of choice for studying such crystalline defects. The understanding of the origin and behavior of structural defects may allow their tailoring and the development of low defect density materials.

III‐nitrides are classically grown along the polar c‐direction. In this case, internal electric fields play a major role in the properties of heterostructures. In order to eliminate or at least to reduce the influence of internal electric fields, growth along alternative directions with the c‐direction in the growth plane (nonpolar) or inclined to it (semipolar) have been developed. Heteroepitaxial nonpolar and semipolar films contain large densities of basal plane stacking faults (BSF) and related partial dislocations together with prismatic stacking faults.

In this presentation, results of TEM studies of semipolar GaN films deposited on patterned substrates will be presented. Different TEM techniques from diffraction contrast classical imaging, high resolution TEM and scanning TEM to analytical techniques as energy dispersive X‐ray spectroscopy (EDS) have been employed to obtain a complete view of semipolar GaN microstructures. The understanding of the nucleation and propagation of defects allowed us to develop several growth processes resulting in a drastic reduction of the defect density:

A 3‐step growth process for (11‐22) GaN deposited on patterned r‐sapphire leads to high quality GaN films with dislocation densities as low as 7x10
7
cm
‐2
and BSF densities below 10
2
cm
‐1
(figure 1)
1,2
.

A method based on the introduction of Si at an intermediate stage of the growth (before coalescence of nucleation islands) allows, in the case of (10‐11) GaN on patterned (001) 7° off‐axis Si, blocking the propagation of dislocations (figure 2). A 4nm thick Si‐rich (5% Si from EDS analysis) layer is revealed by HRSTEM (figure 3). This layer has the wurtzite structure of the surrounding GaN and does not introduce significant strain (as revealed by GPA analysis).

Selective growth on deeply grooved sapphire substrate results in GaN (11‐22) bands with a dislocation density in the mid 10
6
cm
‐2
on 100µm‐wide regions compatible with the fabrication of laser diodes.

Besides presenting results on the improvement of material quality through innovative growth processes, this presentation emphasizes the importance of TEM studies for the developments of heteroepitaxial semiconductors structures.
CelDi
: Development of an advanced solid / fluid reaction stage for
SEM
Authors: Salacroup Johan , Gonnet Gautier , Candeias Antoine , Brau Henri‐Pierre , Szenknect Stéphanie , Ivaldi Paul , Podor Renaud
Published: December 2016

In numerous scientific fields such as life, materials and Earth sciences, or quality controls of industrial processes, there is a growing interest for the direct observation ‐ at the submicroscopic scale ‐ of processes occurring at solid / liquid and solid / gas interfaces. So far, only few experimental cells were designed to address this challenging issue. Most of them are devoted to a specific use in Transmission Electron Microscopy (TEM) and are not suitable for observation of large (or thick) samples and the other cells designed to be used in a Scanning Electron Microscope (SEM) chamber do not allow fluid flow.

To address this issue, a dedicated device was developed according to the following requirements: 1) The sample holder must be suitable for large samples. 2) The device must allow the renewal of the fluid through a continuous flow. 3) The device should be sufficiently efficient and secure to be used in any type of conventional SEM. 4) The device should be easy to implement and user friendly. A first prototype (Fig. 1) was recently tested and patented [1].

For the proof of concept, it was used to perform
in situ
experiments during which series of images was recorded with a SEM, using the back scattered electron detector, high vacuum in the SEM chamber and e‐beam acceleration voltage of 30kV. Fig. 2. presents several images of the growth of NaCl crystals obtained from a supersaturated solution. The image resolution is good enough to see details of a size of 50 nm. The liquid system that is inside the stage remained isolated from the SEM chamber during the complete experiment.

The CelDi project aims at the development of the second generation of this tool, integrating more safety protections, increasing the resolution of the images, finding solutions to achieve an easy and friendly use of the device. It will be possible to integrate a specific and fast BSE detector in combination with the stage, and work is under progress to develop automatized image processing through dedicated software.

In parallel, several tests will be carried out for different scientific applications in material science (corrosion) and life science (observation of live cells) to demonstrate the capabilities of the CelDi device.
In‐situ reduction of platinum alloys using environmental
(S)TEM
Authors: Ward Michael , Boyes Ed , Gai Pratibha
Published: December 2016

Platinum‐cobalt amongst other platinum alloys has desirable properties for use in fuel cells, having higher activities and stability than current commercial catalysts which only use platinum. Research into structures of platinum‐cobalt alloy nanoparticles has greatly benefited from electron microscopy, particularly
Z
contrast HAADF‐STEM imaging which is able to resolve the nanoparticle and surface structure with atomic resolution [1].

Most of the electron microscopy has focused on the characterisation of as prepared particles which have underwent further treatment outside the microscope [1, 2]. These annealing experiments produce ordered structures on the surface and fully ordered cubic (Pt
3
Co) or tetragonal phase (PtCo) alloys depending on the composition and annealing temperatures and times. The studies have been aided by
in‐situ
(S)TEM studies which focus on the segregation of platinum and cobalt is observed inside the microscope at atomic resolution [3, 4]. Although there are many studies, often accompanied by EELS and EDX there appear to be few studies on the initial interaction between platinum and other metals which may be more representative of the activation of a commercial catalyst which is produced in bulk.

Inspired by annealing experiments performed on thin films for magnetic applications [5], we took a different approach by using aberration corrected environmental STEM and a MEMS heating holder to investigate how platinum, cobalt and cobalt oxides interact upon initial annealing. We sputtered thin films of platinum and cobalt to make model systems similar to those used in magnetic thin films. Figure 1 shows a low and high magnification image of the as deposited film. The high magnification image shows mostly platinum crystallography but several amorphous regions and CoO crystals in the background. By annealing
in‐situ
, in hydrogen gas (to remove cobalt oxide, typically used as a cobalt precursor) we were able to form new structures. Figure 2 shows the film shortly after being at 450 °C and 700 °C. In both cases alloyed regions were generally found but only completely ordered particles were observed at 700 °C. The 700 °C annealing temperature produces a good distribution of nanoparticles which can be further explored. Similar experiments are to be carried out on other alloys including platinum‐copper and platinum‐nickel. Results from these experiments have importance to the development of fuel cells and other industrial processes. The behaviour of platinum‐cobalt thin films under annealing may also find use in magnetic film research.
Multi
ADF
detector tomography for
3D
characterization of heterostructures
Authors: Sentosun Kadir , Sanz Ortiz Marta N. , Batenburg K. Joost , Liz‐Marzán Luis M. , Bals Sara
Published: December 2016

Characterization of core‐shell type nanoparticles in 3 dimensions (3D) by transmission electron microscopy (TEM) can be very challenging. Especially when both heavy and light elements co‐exist within the same nanostructure, artefacts in the 3D reconstruction are often present. A representative example would be a particle comprising an anisotropic metallic (Au) nanoparticle coated with a (mesoporous) silica shell. To obtain a reliable 3D characterization of such an object, we collected high angle annular dark field scanning TEM (HAADF‐STEM) and annular dark field tilt series (ADF‐STEM) for tomography (Figure 1A and 1B respectively). Although the series acquired by ADF‐STEM shows both the Au and the SiO
2
, artefacts are clearly present in the 3D reconstruction (Figure 2). Since the observed artefacts may cause loss of information or may lead to misinterpretation, it is extremely challenging to obtain reliable 3D results for core‐shell hybrid materials using conventional electron tomography. When selecting an optimal value for the collection angle, a compromise is needed between optimal contrast, produced by the atomic number of coexisting elements, and the minimization of diffraction contrast.

We here overcome this limitation by exploiting the flexibility of modern TEM instruments that enable one to collect multiple (HA)ADF‐STEM series simultaneously, by using different (HA)ADF detectors at the same time. This multi‐mode approach is very dose‐efficient, as one is able to collect 2 images while keeping the necessary electron dose the same. Tilt series were simultaneously acquired using an ADF detector with inner and outer collection angles of 35 and 125 mrad and a HAADF‐STEM detector using inner and outer collection angles of 150 and 220 mrad, respectively. To remove the artefacts that appear in the ADF‐STEM tilt series, we removed the complete Au nanoparticle from the ADF‐STEM projection images. Next, a technique known as inpainting was applied[1]. This approach replaces the absent information by a continuation of the texture of the surrounding area. This procedure was performed for each projection image of the tilt series separately (Figure 3A).  The processed tilt series was then used as an input for 3D reconstruction using the SIRT algorithm implemented in the ASTRA toolbox[2]. Finally, the 3D HAADF‐STEM and ADF‐STEM reconstructions are combined into one single visualization using the AMIRA software as illustrated in Figure 3B [3]. In this manner, we were able to reliably characterize the structure of mesoporous SiO
2
Au nanoparticles. It must be noted that the methodology we propose here is generally applicable to a broad range of core shell hybrid nanostructures.
Laser triggered microfabricated ultrafast electron beam blanker
Authors: Weppelman Gerward , Moerland Robert , van Tol Ruud , Heerkens Carel , Hoogenboom Jacob , Kruit Pieter
Published: December 2016

Femtosecond electron pulses are typically created by illuminating a flat photocathode with femtosecond laser pulses. [1] However, flat photocathodes have a low reduced brightness, 2 orders of magnitude lower than a Schottky electron source. A higher brightness can be achieved using a cold field emitter illuminated with femtosecond laser pulses. [2] Using a cold field emitter illuminated with UV pulses the group of Zewail has realized an ultrafast SEM. [3] However, such an USEM cannot easily be switched back to continuous beam operation. In addition, the pulse has to be accelerated from the tip onwards which leads to a broadened pulse at the sample.

Here, we propose a beam blanker for use in regular EMs that allows switching between continuous‐beam and ultrafast modes of operation. Previous approaches to ultrafast beam blanking were based on beam blankers using GHz magnetic or electric fields. [4,5] These GHz cavities are still relatively large and can't be inserted directly in a standard commercial SEM.

We use a miniaturized beam blanker controlled by a photoconductive switch, illuminated with femtosecond laser pulses, as schematically depicted in Figure 1. Hence, the blanker is locked jitter‐free to the laser. We show that such a beam blanker needs to have micrometer scale dimensions for ultrafast operation. COMSOL simulation results, including the full 3D blanker design, are used to evaluate the time response of the system.

We fabricated and integrated the deflector plates and the photoconductive switch in a one micrometer‐scale device, see Figure 2. We will show fabrication results of the ultrafast blanker and its incorporation on an insert for a FEI Quanta FEG 200 SEM. We will also show alignment of both laser and electron beam on the ultrafast beam blanker. Also results will be presented showing laser triggered deflection of the electron beam.
Element partitioning and atom location of alloying elements in
Co
‐base superalloys
Authors: Wang Li , Oehring Michael , Lorenz Uwe , Pyczak Florian
Published: December 2016

The discovery of an ordered L1
2
precipitate (Co
3
(Al, W)) phase in the ternary Co‐Al‐W system in 2006 which is stable up to temperatures of 950 °C attracts significant research on microstructure design for a new class of load‐bearing Co‐base high‐temperature alloys. The low mismatch between the fcc γ‐Co solid‐solution phase and the L1
2
γ'‐Co
3
(Al, W) phase helps to establish a microstructure with coherent cuboidal γ' precipitates embedded in a continuous γ matrix, analogous to Ni‐base superalloys. These new Co‐base superalloys have the potential to exhibit excellent high‐temperature mechanical properties and are considered to be ideal turbine blade materials.

In the ternary Co‐Al‐W system, it is reported that the L1
2
phase is in equilibrium with the B
2
‐CoAl and D0
19
‐Co
3
W phases and transforms into those after extended annealing
[1]
. In order to increase the temperature capability and stabilize the γ'‐L1
2
phase, additional alloying elements are added to the Co‐Al‐W system. With additions of Cr, Mo, Ni, Re, Ta and V, the solidus and liquidus temperatures of Co‐Al‐W alloys are 100‐150 °C higher than those of advanced Ni‐base single‐crystal alloys strengthened by the L1
2
phase
[2]
. It is speculated in literature that Mo, V, Nb, Ta and Ti increase the γ' solvus temperature of Co‐Al‐W superalloys because they occupy B‐sites in the A
3
B ordered γ' phase and thereby increase its volume fraction, while Fe, Mn and Cr tend to distribute to the γ phase and decrease the amount of γ' phase
[3]
. Because the distribution of additional elements will influence the morphology and amount of the γ' phase, it is important to investigate their partitioning behavior and atomic site occupation in the Co‐Al‐W system. Therefore the high‐temperature strength and thermal stability of Co‐base superalloys are optimized.

In this study, alloy samples with nominal compositions Co‐9Al‐9W‐2X (X=Ti, Nb, V, Ta, Cr and Mo, at.%) were produced. The partitioning behavior of the alloying elements between γ and γ' phases were investigated by energy‐dispersive X‐ray spectroscopy (EDS) in the transmission electron microscope (TEM). In order to analyze the element occupation at atomic sites, atom location by channeling enhanced microanalysis (ALCHEMI) technique was applied.

The details about the partitioning behavior and the atomic site occupation of alloying elements will be discussed in the presentation.
Cryo‐electron microscopy structure of La Crosse orthobunyavirus polymerase in presence or absence of viral
RNA
Authors: Malet Helene , Gerlach Piotr , Reguera Juan , Cusack Stephen
Published: December 2016

Bunyaviridae
is the largest family of segmented negative strand viruses (sNSV) which also include
Orthomyxoviridae
and
Arenaviridae
. Central to their viral cycle is the RNA‐dependent RNA polymerase which replicates and transcribes the genome segments within circular ribonucleoprotein particles (RNPs). Here we describe a cryo‐electron microscopy reconstruction of the full length La Crosse polymerase in complex with viral RNA (Figure 1), together with a reconstruction of its apo truncated form (Δ‐Cterminal construct, Figure 2). Combined with the X‐ray structure determined in the group, we provide a partial pseudo‐atomic model of La Crosse polymerase (Figures 1 and 2). Identification of distinct template and product exit tunnels (Figure 3) and structural analysis of RNP (Figure 4) allows proposal of a detailed model for template‐directed replication with minimal disruption to the circularised RNP. The similar overall architecture and vRNA binding of monomeric LACV to heterotrimeric influenza polymerase, despite high sequence divergence, suggests that all sNSV polymerases have a common evolutionary origin and mechanism of RNA synthesis.
Precise
STEM
measurement of defocus and aberration in monolayer graphene
Authors: Sawada Hidetaka , Kirkland Angus
Published: December 2016

Improvements to the Z height resolution in a scanning transmission / transmission electron microscope (S/TEM) are important for 3D reconstruction of local specimen topography and for quantitative image analysis using comparisons of experimental images to simulations. The lateral position perpendicular to the direction of the electron trajectory in aberration corrected S/TEM instruments has a sensitivity comparable to most atomic spacing which can be directly determined in experimental images to an accuracy of a few pm. However, the resolution in the Z direction parallel to the electron trajectory is of the order of only a few nm as determined by direct experimental measurement using confocal electron microscopy of extended objects [1] or in HAADF STEM images of point like objects [2]. This resolution is an order of magnitude larger most atomic spacing and therefore to accurately determine Z height information at atomic resolution, a method for precise measurement of the specimen height is required.

Toward obtain better accuracy for Z direction measurements, we have used the lateral magnification change of an ideal specimen of monolayer graphene, where in reciprocal space with a probe forming mode, the magnification of the lattice depends on the value of the defocus. Ronchigrams have been recorded with a focused STEM probe of atomic dimensions at a large camera length of 80 cm at 80 kV [Fig. 1(a)]. The Ronchigram was divided into local angular areas to analyze the local magnification dependent on the probe‐forming aberrations. Importantly the lattice parameter of graphene is accurately known and changes in the lattice pattern with beam tilt or specimen tilt are smaller than for other, thicker crystalline specimens. The local magnification in each local area was analyzed using an auto‐correlation function [Fig.1(b)] [3]. Six peaks surrounding the center peak of the auto‐correlation function from a graphene image were identified and were fitted with an oval [Fig.1(c)]. Using the fitted parameters describing the oval shapes at local angular areas, the distance between the probe and the graphene monolayer (defocus) was measured precisely to be ‐ 58.52 nm. Since the lattice parameter of a specimen is known, only one Ronchigram pattern is required to derive the value of defocus. Other aberration coefficients for the probe forming lens also can be calculated using this method [Table. 1].
Structure of short period
In(Ga)N/GaN
superlattices comprising ultra‐thin quantum wells
Authors: Dimitrakopulos George , Bazioti Calliope , Karakostas Theodoros , Kioseoglou Joseph , Pavloudis Theodoros , Kret Slawomir , Koziorowska Julita , Suski Tadek , Dimakis Emmanouil , Moustakas Theodore , Komninou Philomela
Published: December 2016

InGaN quantum wells (QWs) grown along the polar c‐axis are currently the principal structural elements of III‐nitride optoelectronic device active regions. Further advancement of their operational wavelength range with high internal quantum efficiency in the green part of the spectrum may be facilitated through the use of short period In(Ga)N/GaN superlattices comprising QWs with a minimal number of monolayers (MLs). At the same time, ultra‐thin InN/GaN QWs have recently generated considerable interest, due to the theoretical prediction that they may exhibit topological insulator properties [1], thus opening up new prospects for applications in quantum computing and spintronics. In recent work, two‐dimensional electron gas properties were demonstrated in ML‐thick, nominally InN QWs, as well as a temperature‐independent behavior in the diagonal resistance, indicating the topological nature of the 2DES [2].

We have studied a series of In(Ga)N/GaN short period superlattice (SPS) heterostructures deposited at low growth temperatures. The examined samples comprised SPS with nominally 1, 2, and 4 ML QW thicknesses grown by molecular beam epitaxy (MBE) on (0001) GaN/sapphire MOVPE templates. High resolution transmission electron microscopy (HRTEM), and high resolution high‐angle annular dark field imaging in scanning TEM mode (HRSTEM) have been employed in order to ascertain the compositional homogeneity due to issues such as the desorption, clustering, and diffusion of indium into the GaN barriers. Such issues may be aggravated by the large elastic strain associated with the pseudomorphic growth of the ultrathin QWs. On the other hand, it is also possible that the stress due to the misfit may in fact stabilize the growth of InN at unusually high temperatures. Fig. 1 illustrates HRTEM and HRSTEM images of a 1 ML InN / 10 ML GaN SPS and of a 4 ML‐thick QW. Strain and compositional mappings were implemented by using peak finding with the peak‐pairs method, and geometrical phase analysis. Z‐contrast image calibration was performed using reference samples and image simulations.

The results were compared with energetic calculations of pertinent supercells using both molecular dynamics with a modified Tersoff interatomic potential, and ab initio density functional theory. In both cases, deviation from the biaxial stress state of InN was identified for these QWs in agreement with the experimental observations.
Spin‐multislice simulation of an electron inside the objective lens of a TEM
Authors: Grillo Vincenzo , Schachinger Thomas , Karimi Ebrahim , Schattschneider Peter
Published: December 2016

Spin filtering of an unpolarized beam in a TEM is a fascinating field of research. Bohr conjectured that it is impossible to spin filter an electron beam or, using Bohr words, “to observe the spin of the electron, separated fully from its orbital momentum, by means of experiments based on the concept of classical particle trajectories”[1].

However, the principle seems to be violated by theoretical calculations [2,3]. One of the most convincing proposals for free electron polarization is a multipolar Wien filter. But the fields involved are typically very large [2] while multipolar Wien filters in microscopy are still rare. The device, together with the diffractive elements, is called as “q‐filter” where q hints at the topologic charge of the field.

The objective lens of the microscope provides a very large field with the potentiality of introducing a spin‐orbit coupling, we performed spin‐multislice simulations [4], where a Bessel beam was propagated through the objective lens (modeled as a Glaser field) in order to quantify the degree of spin polarization.

We will discuss in particular that the spin‐orbit conversion in the pre‐ and post‐field can be understood in terms of the q‐filter.

Fig 1 a shows on the left a scheme of the objective lens and of the electron wavefunction (blue) passing through it. A schematic “Bohmian” trajectory is indicated by a curve. The image also features arrows indicating the classical spin orientation along the curve for an initial state with spin |↑> along the optic axis.

The fig 1b indicates the multislice calculated evolution of the wavefunction. While the expectation value of the spin operator S has components < S
x
>=< S
y
> =0 we can track the expectation of the x,y vector P=(S.r,S.t) (with r being the in plane position versor and t its orthogonal in plane versor). The result is shown in fig 2. P represents a sort of local in plane projection of the spin operator. To a good degree of approximation here |P| is equal to the rate of conversion from |↑> to |↓>.

The results indicates a net, typically weak , increase of P as an effect of the objective lens .

The overall final wavefunction is described in Fig 3 as a non‐separable spin‐orbital angular momentum state.

The multislice results are in quantitative agreement with ray tracing calculations, confirming the reliability of both methods in this case. However, the multislice approach enables us to use less classical states like Laguerre Gauss beams, to explore possible advantages and more quantum physical effects.
Electron holography by means of tilted reference waves
Authors: Röder Falk , Lubk Axel , Houdellier Florent , Denneulin Thibaud , Snoeck Etienne , Hÿtch Martin
Published: December 2016

Off‐Axis Electron Holography permits the direct reconstruction of amplitude and phase of electron waves elastically scattered by an object (see,
e.g
., [1]). The technique employs the Möllenstedt biprism to mutually incline an object modulated wave and a plane reference wave to form an interference pattern at the detector plane. Limited coherence of the electron beam in presence of aberrations attenuates high spatial frequencies of the object exit wave spectrum, which is illustrated by the sideband envelope function for a non‐corrected TEM in Fig. 1a.

In this work, we explore an extension of the conventional setup given by deliberately tilting the reference wave independent from the object wave. This allows the transfer of spatial frequencies beyond the conventional sideband information limit in Fig. 1a as predicted by a generalized transfer theory for Off‐Axis Electron Holography [2]. This is because a reference wave tilted by

q
0

compensates the aberration impact on the spatial frequency

q
0

of the object wave spectrum. The resulting transfer envelope for a tilt of

q
0x

= ‐10/nm perpendicular to the post‐specimen biprism is shown in Fig. 1b, where the contrast maximum of the total envelope (TCC) is located at

q
0x

. Thus, an off‐axis hologram series with varying reference wave tilt allows in principle a linear synthesis of an effective coherent aperture with a radius reaching out beyond the conventional information limit. Furthermore, an object‐independent measurement of aberrations as well as dark‐field electron holography can be realized using this setup.

The experimental realization of an arbitrarily tilted reference wave is challenging and could be realized for the first time at the Hitachi HF3300C I2TEM at CEMES Toulouse for one direction [3]. We used an additional biprism placed in the illumination system. Three condenser lenses were adjusted to provide a demagnified image of the condenser biprism at the sample plane under parallel illumination (Fig. 2). The pre‐specimen deflectors were adapted to maintain the incident wave vector of the object wave and to realize a tilt of the reference wave as a function of the condenser biprism voltage. Optimal condenser lens settings were found by means of paraxial ray tracing (Fig. 3) finally producing a mutual tilt of up to 20/nm at the object plane. We verified the kink‐like phase modulation of the incident beam by means of holographic measurements. Contrast transfer theory including condenser aberrations and biprism instabilities was applied to explain detailed fringe contrast measurements. Finally, we have experimentally shown that dark‐field holography [4] can be conducted with an object‐independent reference.
In Situ
TEM
Study of Fatigue Crack Growth of
Cu
Thin Films Using a Modified Nanoindentation System
Authors: Bufford Daniel , Stauffer Douglas , Mook William , Asif S.A. Syed , Boyce Brad , Hattar Khalid
Published: December 2016

Material fatigue is often the limiting factor for many engineering cases. Repeated cyclic loading, even at stresses well below the monotonic yield stress of the material, leads to the accumulation of microstructural damage, crack initiation, crack growth, and eventual failure of the device. In terms of the number of loading cycles, the high cycle regime of >10
4
loading cycles is often of interest, although there are cases in which fatigue lifetimes may reach >>10
7
cycles. Cyclic loading experiments with bulk specimens can determine probabilistic fatigue lifetimes, however, a more fundamental understanding of the crack initiation and growth regimes is desired. These, early stage I, fatigue processes are by their very nature of limited size. Therefore, in situ nanomechanical testing in the transmission electron microscope is a good match in terms of both size and time scales. However, to date no general capability for fatigue loading has been made available. For this set of experiments, an in situ nanomechanical system from Hysitron, Inc. was modified to use a sinusoidal loading function, capable of frequencies from 1 to 300Hz, where the resulting displacement amplitude and phase is measured using a lock‐in amplifier. Thus it is now possible to reach the 10
6
cycle threshold within one hour. Here, observations of microstructural changes near propagating cracks in nanocrystalline Cu are presented.

Magnetron sputtered Cu films deposited on single crystal, then floated onto Push‐to‐Pull MEMS devices. These devices provide a micromechanical test frame converting an indentation into a tensile force. Focused ion beam milling was employed to remove excess film, leaving a tensile specimen [1]. Mechanical loading experiments were performed at frequencies from 1 to 200 Hz with mean loads of ≈100 µN, and load amplitudes of ≈50 µN. The total number of accumulated cycles typical exceeded 10
5
cycles. Local microstructural change was characterized by bright field TEM video during loading. Testing was periodically paused to collect additional still micrographs, as well as data collection by a precession electron diffraction‐assisted automated orientation mapping technique [2]. Observable changes in contrast preceded crack nucleation. These cracks then stably propagated prior to rapid unstable failure. Localized microstructural changes occurred near the propagating crack including larger grains oriented differently from the surrounding matrix, figure 1, indicative of possible fatigue‐induced grain growth [3].
Multi‐modal in vivo visualization of single cell dynamics by
1P
,
2P
, light‐sheet, and on‐chip technologies
Authors: Nishimura Satoshi
Published: December 2016

We made fluorescence multi‐scale imager including 1P, 2P, light‐sheet, microscope, and on‐chip sensors for visualization of XYZT single cell dynamics in vivo. We integrated five microscope for systematic evaluation of living animals.

First visualization system is super‐resolution imaging based on non‐linear optics, with X‐ (resonance), Y‐(galvano), and Z‐(piezo) axis scanning. Real‐time, multi‐color XYZT multi‐photon imaging enabled us to visualize single blood cell behavior in vessels and stroma. Spatial and time resolution was improved by pattern illumination. We analyzed thrombus formation, and inflammatory responses under microscope. We developed thrombus formation animal models, and elucidated cellular mechanisms of cardiovascular diseases. We directly manipulated cells by photo‐chemical reactions, and two photon lasers to induce and observe thrombotic reactions. Using this system, we elucidated contribution of endothelial injuries to thrombus formation.

Second, high‐resolutions and broad‐imaging field was simultaneously enabled using 8K CMOS technologies, and 1P spinning disk confocal. We visualized whole organs and single cell in one image, and revealed complicated cell‐cell interactions networks in single view. 8K, 60fps, and multi‐color imaging visualized single platelet dynamics and tissue structural changes in single image.

Third, we performed light‐sheet imaging for living mice, and enabled high‐speed observation of living animals.

Fourth, macro imaging system for awake mice, pigs and humans was also developed, and free behavior monitoring revealed the tight association between metabolism and vascular reactions during daily life. Bioluminescent and fluorescent imaging from body surface using CMOS camera, image intensifier, and macro‐lens enabled us to visualize cellular dynamics without anesthesia.

Last, wearable, implantable, and minimized devices for non‐invasive recording were also developed using lens‐less and on‐chip technologies. We utilized SCMOS, micro‐lens array, and LED illumination technologies.

In sum, we developed multi‐scale imaging system which can evaluate cellular networks dysregulations in diseased conditions.
Structural basis of Nanobody‐mediated plant virus resistance and vector transmission revealed by cryo‐
EM
Authors: Hemmer Caroline , Orlov Igor , Ackerer Léa , Marmonier Aurélie , Hleibieh Kamal , Schmitt‐Keichinger Corinne , Vigne Emmanuelle , Gersch Sophie , Komar Véronique , Belval Lorène , Berthold François , Monsion Baptiste , Bron Patrick , Lemaire Olivier , Lorber Bernard , Gutiérrez Carlos , Muyldermans Serge , Demangeat Gérard , Klaholz Bruno , Ritzenthaler Christophe
Published: December 2016

Since their discovery, single‐domain antigen‐binding fragments of camelid‐derived heavy chain‐only antibodies, also known as Nanobodies (Nbs), have proven to be of outstanding interest as therapeutics against human diseases and pathogens including viruses, but their use against phytopathogens remains limited. Many plant viruses including Grapevine fanleaf virus (GFLV), a vector‐transmitted icosahedral virus related to animal picornaviruses, have worldwide distribution and huge burden on crop yields representing billions of US dollars of losses annually, yet solutions to combat these viruses are often limited or inefficient. Here we show that a Nb specific to GFLV confers strong resistance to GFLV upon stable expression in plants due to virus neutralization at an early step of the virus life cycle, prior to cell‐to‐cell movement. To address the involved molecular mechanisms, we determined the cryo‐ electron microscopy (cryo‐EM) structure of the Nb‐virus complex at 2.8 Å resolution revealing the stoichiometric 1:1 complex formed with the viral capsid protein (CP). Remarkably, the conformational surface epitope recognized by the Nb covers a cavity analogous to the canyon of picornaviruses implicated in receptor binding. By showing that this cavity is also involved in nematode transmission of GFLV, our results suggest a structural conservation driving vector specificity and receptor binding among plant and animal Picornavirales. Our findings will not only be instrumental to confer resistance to GFLV in grapevine but more generally they pave the way for the generation of novel antiviral strategies in plants based on Nbs.
Mg
‐calcite formation in a freshwater environment (Lake Balaton): nucleation, growth, structure and composition
Authors: Pósfai Mihály , Nyirő‐Kósa Ilona , Rostási Ágnes , Bereczk‐Tompa Éva , Cora Ildikó , Koblar Maja , Kovács András
Published: December 2016

Lake Balaton can be regarded as a large scientific laboratory in which many interesting aspects of carbonate mineral formation can be studied, with relevance to the general understanding of nanoscale processes that govern crystal nucleation and growth in a natural aqueous system. The lake is extremely shallow for its size (on average 3.5 m deep and 70 km long), its calcareous water Mg‐rich (with a Mg/Ca molar ratio ranging from (~1 to 4), typically displaying a chemical gradient along the W–E long axis of the lake, as a result of the main inlet and outlet being located at opposite ends. The bottom sediment is a soft grey mud, with 30 to 80% of it consisting of Mg‐calcite, a mineral that precipitates from the water (Fig. 1a). We used SEM, TEM and STEM techniques to characterize this Mg‐calcite, in order to obtain a better understanding of its formation and role in the ecosystem.

Freshwater calcite is known to nucleate on biological material, primarily on picoplankton (cyanobacterial) cells. In contrast, the Mg‐calcite in Lake Balaton is closely associated with few‐unit‐cell‐thick stacks of clay (smectite) layers (Figs. 1b and c), suggesting that it either nucleated on smectite or adsorbed to the clay flakes. Our laboratory experiments confirm that adding smectite to filtered lakewater dramatically induces Mg‐calcite precipitation; thus, the nm‐scale clay fragments likely serve as nucleation sites. Since the lake sediments are stirred up by even gentle winds, most of the time smectite particles are readily available for Mg‐calcite nucleation. A special case occurs when the lake is frozen and even the clay particles can settle: only bacterial cells are available as nucleation sites (Fig. 2a), and encrustation of cells results in tube‐shaped, porous Mg‐calcite particles (Fig. 2b).

Mg‐calcite that formed under „normal” conditions (i.e., nucleated on smectite) typically occurs in the shape of elongated, several μm‐large, aggregate‐looking particles (Fig. 2c). Even though they appear to be composed of many smaller crystals, SAED patterns suggest that the particles are perfect single crystals (Fig. 2d). The single crystalline nature of the particles probably results from a dissolution/reprecipitation process that preserves the original shapes of particles.

The Mg content of the calcite varies from 2 to 20 mol%, depending on the water budget (dilution) and geographical location in the lake. Mg‐calcite that nucleated on bacterial cells is highly enriched in Mg; according to SAED patterns, Mg and Ca ions do not order in the structure. Our results provide new information on freshwater calcite nucleation and on the biologically assisted precipitation of high‐magnesian calcite.
Light and electron microscopic evaluation of lattice corneal dystrophy (
LCD
)
Authors: Seckin Ismail , Cicik Mustafa Erdogan , Arslan Osman Sevki , Isildar Basak , Doğan Cezmi , Burgazdere Ozan , Hagverdiyeva Samira
Published: December 2016

Introduction

There are three types of corneal dystrophies: anterior, posterior and stromal. Lattice corneal dystrophies (LCD) are a part of stromal dystrophies and characterized by an accumulation of amyloid deposition with in the stroma. LCD is diognosed with biomicroscopic examination and the diognosis confirmed by histological examination after keratoplasty. Genetic analysis may be required for specific cases.

In our study, the corneal buttons of four patients, three of them belonging to the same family, who applied to the Ophthalmology Clinic of Cerrahpasa Medical Faculty with lattice corneal dystrophy, histologically examined with light and electron microscopies.

Material‐Method

For light microscopic examination, a portion of corneal buttons were embedded into the parafine blocks after fixation and dehidration steps. Parafine blocks were cut as 5 µ sections and stained with hematoxylen & eosin and periodic acid & Schiff (PAS). For electron microscopic examination, the other pieces of corneal buttons were fixed glutaraldehyde and osmium tetroxide (OsO
4
). After the dehidration, corneal buttons embedded into araldite and the blocks were sectioned 500‐700Å. The sections taken on copper grids were analyzed and photographed.

Findings

In the light microscopic examination of hematoxylin & eosin stained corneal buttons; the local deposits seen in a circular arrangment, was observed and it was suggested that these were amyloid deposits. In only one of the four patients' corneal buttons, these deposits were seen in the stroma, but in the other three patients', belonging to the same family, deposits increased epithelial level and led to significant damage to the epithelium. When the samples were stained with periodic acid‐ Schiff reagent, positive reaction was observed in these deposits. Electron microscopic examination revealed that typical amyloid fibrils also occurred in deposits.

Despite local areas were damaged due to these amyloid deposits, no inflammation was observed.

Conclusion

As in other corneal dystrophies, a systematic approach and histopathological evaluation, including special staining and ultrastructural studies, are critical to a correct diagnosis, also in LCD. Therefore, it is important to make a histopathologic examination for all grafts clinically diagnosed or undiagnosed.
Serial section
(S)TEM
tomography of prokaryotic cells
Authors: Rachel Reinhard , Heinz Veronika , Flechsler Jennifer , Witzgall Ralph , Heimerl Thomas
Published: December 2016

Cell complexity in prokaryotic cells, whether Bacteria or Archaea, is generally considered low, with a single compartment. A few examples, however, have been recently reported showing evidence for compartmentalization [e.g. 1,2,3]. These studies gained in particular from improved sample preparation methods (plunge freezing or high‐pressure freezing), adequate follow‐up methods, and 3D imaging of the samples, either by FIB‐SEM or by electron tomography. Here we report on our analyses of the ultrastructure of exceptional Archaea of the genera Ignicoccus and Pyrococcus, which show remarkable physiological characteristics (e.g. optimal growth at T~85 up to 100°C, under anoxic conditions), and unusual ultrastructural features. Although prokaryotes, these cells have a diameter of > 1 µm, which does not allow for reasonable cryoTEM observation without sectioning. Thus, samples were harvested by gentle concentration on ultrafiltration membranes, cryo‐immobilized by high‐pressure freezing, freeze‐substituted, and finally resin‐embedded; this enabled us to perform 3D ultrastructural analysis by (S)TEM tomography and immuno‐localization studies on sections of the same resin block.

So far, we are about to routinely investigate single or serial sections (ranging from 200 up to 800 nm in thickness), on a transmission electron microscope operated as TEM or STEM at 200 keV. For STEM, the illumination is adjusted to have an almost parallel beam, i.e. small convergence angle, resulting in an extended depth of focus [4]. Under these conditions, if the sample is imaged at high tilt angle (e.g. up to 70 deg), all visible image details are seen ‘in focus’, which greatly facilitates image alignment during 3D image reconstruction using IMOD (Boulder, Co, USA). In addition, the system in use allows parallel acquisition of BF and DF signal. Reconstruction and visualization is done using either WBP or SIRT in IMOD, and final manual segmentation using AMIRA®.

Our 3D data show novel and remarkable ultrastructural features: (A) Ignicoccus cells possess two membranes; in the intermembrane compartment, there is a complex endogenous membrane system. Attached to the outer cellular membrane, we have localized enzyme complexes for proton gradient generation, ATP and acetyl‐CoA synthesis, and CO
2
fixation [5]. (B) Pyrococccus cells possess flagella of the archaeal type [6], i.e. extracellular filaments involved in motility and cell‐cell interaction, and a cytoplasmic membrane and an S‐layer, and a so far unknown layer inside the cytoplasm presumbably involved in anchoring or organizing the flagellar bundle.
In‐situ observations of nanoparticles of carbon supported copper electrocatalyst during electrochemical cycling
Authors: Pacaud Jerome , Demortiere Arnaud , Dachraoui Walid , Sahin Niat Ege , Habrioux Aurelien , Comminges Clément , Napporn Tekko , Kokoh Boniface , Aguy Stephane
Published: December 2016

Microwave assisted synthesis of metallic nanoparticles is an attractive way for the elaboration of electrocatalysts using energy efficient processes. This method consists in the use of a weak reducing agent that acts as solvent with high boiling point at the same time. Among the possible solvents, diethylene glycol is one of the most suitable molecules for microwave absorption and heating. Since microwave energy leads to a more uniform heat‐treatment than a conventional heating process, the overall activation energy for metal ion reduction decreases under microwave exposure. The carbon supported copper catalyst described herein is a good candidate for electrocatalytic processes due to the fact that copper is an abundant and effective transition metal for a number of reactions.

Observations were conducted in a Tecnai G2 fitted with a FEG, an EDX spectrometer and GIF Quantum. STEM‐HAADF was the primary technique of image recording. Scan speed was kept high in order to follow the kinetic of the evolution of the system. In this experiment, we use a sealed TEM cell recently developed by Protochips , i.e. liquid/bias TEM specimen holder (Poseidon 510). This system permits to study materials under electrochemical environment in TEM instrument. The sample (Copper nanoparticles on carbon) that is inside the sealed TEM cell (liquid gap is from 500 nm to 3000 nm) is immersed in the liquid electrolyte introduced by micro‐fluidic system (here aqueous electrolyte KCl/H2O at 0.01ML) and then electrochemically cycled using designed working electrode (glassy carbon) on transparent SixN1‐x windows. For electrochemical measurement, the applied currents were between 100 nA and 500 nA. Voltammogram was taken to ensure the correct oxidation and reduction of the copper atoms during a cycle. A strong corrosion of the supporting carbon is then observed. Experiments at constant current (chronopotentiometry) show the coalescence of large Cu grains and their disappearance depending on the applied current (See figure 1 and 2). Gas bubble formation is also observed (Figure 3). In these specific liquid experiments, optimized conditions remain to be found for TEM observation and in‐situ cycling, especially for electrocatalysis which is particularly demanding in terms of control of the experimental condition.