bluuebunny/crossref_metadata_2025 huggingface dataset:
Application of
FIB‐SEM
tomography for analyses of ceramic coatings deposited on titanium alloy and material‐cell interface
Authors: Karbowniczek Joanna , Gruszczyński Adam , Kruk Adam , Czyrska‐Filemonowicz Aleksandra
Published: December 2016

Biomedical engineering is a fast developing field of medicine, employing biomaterials to replace or augment damaged and diseased tissues. Growing population with osteoarthritis problems and extending life span contributes to increased demand for effective solutions in arthroplasty. Titanium alloys are the materials of the choice in case of elements of artificial joints. They are implanted directly into the bones. Long lasting performance of those implants depends on their integration with surrounding tissues. Surface modification of titanium alloys is performed to improve osseointegration. It can be achieved by deposition of porous, ceramic coatings. Such a structure facilitates bone cells ingrowth and stable anchorage of the implant in the bone. Understanding of the reactions at the interface between materials and cells will help to optimize the design of new implants with improved performance.

In current work, a titanium alloy (Ti6Al7Nb) was used as a substrate in micro‐arc oxidation (MAO) process. As the effect of this electrochemical reaction the protective and bioactive coating composed of titanium dioxides, calcium titanate and calcium phosphates was formed on the metallic sample. The obtained complex ceramic, porous layer was investigated by SEM and SEM‐EDS (Merlin Gemini II, Zeiss) and FIB‐SEM tomography (NEON CrossBeam 40EsB, Zeiss) to analyze microstructure, chemical composition and internal structure, including pores network.

In vitro
cell culture is a standard method to evaluate biocompatibility of new or modified materials. Thus, osteoblast‐like cells line MG‐63 were cultured for three days at the surface of coated titanium alloy samples. Subsequently cells attached to the ceramic coating were fixed, dehydrated and gold sputtered for electron microscopy investigations. Cells shape and distribution at rough and porous structure of the coating containing hydroxyapatite (HA) in the outer layer was investigated based on plain view SEM images (Fig. 1a). Area prepared for tomography by FIB milling of surrounding material is presented at Fig. 1b (marked by red square). FIB‐SEM tomography enables to see both material surface and cells at nanometer scale (Fig. 1c). The 3D reconstruction allows for visualization and investigations of the interface between coated material and cell in the analyzed sample volume.
Three‐dimensional core‐shell ferromagnetic nanowires fabricated by focused electron beam induced deposition
Authors: Pablo‐Navarro Javier , Magén César , María De Teresa José
Published: December 2016

Functional nanostructured materials often rely on the combination of more than one material to confer the desired functionality or an enhanced performance of the devices. One of the challenges for Focused Electron Beam Induced Deposition (FEBID) technology is the fabrication of three‐dimensional (3D) heterogeneous nanostructures which can be applied in magnetic memories, logic and sensing [1]. A novel procedure to create nanoscale heterostructured materials in the form of 3D core‐shell nanowires by FEBID technologies has been developed. This new approach has been applied to synthesize standing nanowires with cylindrical metallic ferromagnetic cores of Co‐ and Fe‐FEBID (less than 100 nm thick) coated with a protective Pt‐FEBID shell ranging 10‐20 nm of uniform thickness, using Co
2
(CO)
8
, Fe
2
(CO)
9
and CH
3
CpPt(CH
3
)
3
precursor gases. This architecture aims at minimizing the degradation of magnetic properties caused by the natural surface oxidation of the core to a non‐ferromagnetic material. This is a key issue in such thin ferromagnetic objects with a high surface‐to‐volume ratio [2].

The structure, chemistry and magnetism of FEBID nanowires with cores of Co and Fe have been characterized in Pt‐coated and uncoated nanostructures. Scanning transmission electron microscopy (STEM) imaging and electron energy loss spectroscopy (EELS) experiments have revealed that the surface oxidation is suppressed from the magnetic cores and confined to the Pt layer, while keeping the cylindrical shape of the nanowire (Figure 1). Local magnetic states of uncoated and coated Co‐ and Fe‐FEBID nanowires in remanence have been obtained by off‐axis Electron Holography (EH). After substraction of the phase contribution of the mean inner potential and thanks to the cylindrical shape of the cores, quantitative magnetic induction maps have been obtained which demonstrates that the average magnetization of the ultrathin coated cores is strengthened up to 30% in the thinnest nanowires (50‐nm‐thick cores) with respect to unprotected ones (Figure 2).
The near‐atomic resolution cryo electron microscopy structure of the lactococcal siphophage 1358 virion mature full capsid
Authors: Orlov Igor , Klaholz Bruno
Published: December 2016

Lactococcus lactis, a Gram(+) lactic acid‐producing bacterium used for the manufacture of several fermented dairy products, is subject to infection by diverse virulent tailed phages, leading to industrial fermentation failures. Despite the availability of several antiphage measures, new phages keep emerging in this ecosystem. This constant viral risk has led to a sustained interest in the study of their biology, diversity, and evolution. Lactococcal phages now constitute a wide ensemble of at least 10 distinct genotypes within the Caudovirales order, many of them belonging to the Siphoviridae family. Lactococcal siphophage 1358, currently the only member of its group, displays a noticeably high genomic similarity to some Listeria phages as well as a host range limited to a few L. lactis strains. These genomic and functional characteristics stimulated our interest in this phage.

Here, we report the near‐atomic resolution cryo electron microscopy (cryo‐EM) structure of the 1358 virion mature full capsid. Recent advances in cryo‐EM have enabled structure determination of macromolecular complexes at near‐atomic resolution. However, structure determination and atomic modelling still remains a challenging task susceptible to model bias and overfitting especially while using
de novo
methods. This study demonstrates a practical approach to obtain a rigorously validated atomic resolution cryo‐EM structure allowing to complement the previously published complete structural picture of a unique lactococcal phage at lower resolution and have deeper insight into structural organization of it's capsid.
Study of disorders in zeolite
ITQ
‐39 using structure projection reconstruction from through‐focus series of
HRTEM
images
Authors: Kapaca Elina , Willhammar Tom , Wan Wei , Zou Xiaodong , Moliner Manuel , Martinez Cristina , Rey Fernando , Corma Avelino
Published: December 2016

The structure of an aluminosilicate ITQ‐39 has been determined by electron crystallography, it is one of the most complex zeolite ever solved [1]. ITQ‐39 has a 3‐dimensional channel system with intersecting 10‐ and pairwise 12‐ring channels. It has a highly faulted structure and contains stacking disorder along [100], twinning along [010] and point defects. High‐resolution transmission electron microscope (HRTEM) images were chosen to study these disorders. In order to improve HRTEM data quality, a through‐focus series of 20 HRTEM images were collected and a structure projection image was reconstructed (see Fig. 1) by a contrast transfer function (CTF) correction algorithm using the software QFocus [2]. The through‐focus series was acquired with a constant focus step (‐53.3 Å) and the two‐fold astigmatism was the same through the series. Defocus values and the two‐fold astigmatism were then determined for all the images. CTF correction was performed on each image and the final reconstructed image was obtained by averaging all 20 CTF‐corrected images (see Fig. 1b). Defocus determination revealed that the image series passed through the Scherzer focus condition and contained at least one image close to the Scherzer condition (Fig. 1a). By using structure projection reconstruction the noise in the images was significantly reduced and the structure and pore system were more obvious after the reconstruction. One big advantage is that there is no need to spend time to adjust to a certain focus (e.g. close to Scherzer focus), therefore the acquisition of HRTEM images can be done much faster which is very useful for beam sensitive samples.
A simple shortcut for observing unroofed cells by either
TEM
or
SEM
Authors: Franck Agathe , Lainé Jeanne , Bitoun Marc , Frébourg Ghislaine , Trichet Michaël , Vassilopoulos Stéphane
Published: December 2016

The “unroofing” technique has been successfully used to observe the cytoplasmic side of the plasma membrane (PM) using either light or electron microscopy. Combined to transmission electron microscopy (TEM), it is an invaluable method to reveal the composition of the PM and to directly observe macromolecular complexes including the cytoskeleton and endocytic membrane invaginations. This method has been optimized over decades to preserve membranes close to their native states by the combination of quick freezing of exposed membranes, followed by deep etching and rotary replication (the so‐called “QF‐DE‐RR” technique). However, a serious setback in implementing unroofing combined with QF‐DE‐RR stems from the necessity to use complicated apparatus, such as quick freezing and freeze‐fracturing devices, along with strong expertise to handle them. Moreover, the technical complexity renders these techniques time consuming and reduces the number of samples that can be processed simultaneously.

Here, we present a simple and straightforward protocol for observation of the cytoplasmic side of plasma membrane which only requires chemical treatment of samples prior to replication This method has been optimized towards sample preparation at room temperature, chemical fixation, dehydration, solvent drying and sequential metal coating. Moreover, this technique is easily amenable to higher throughput. We compared either TEM or high resolution SEM analysis of unroofed membranes from adherent cells and show the advantages and disadvantages of each technique towards visualization of the cytoskeleton and different endocytic structures such as clathrin coated pits and caveolae.
The Effect of Vitamin
E
on Ovarian Damage Caused by Nicotine Administration
Authors: Sezer Zehra , Yılmaz Tuğba Ekiz , Güngör Zeynep , Guzel Elif
Published: December 2016

Nicotine causes the formation of free radicals and oxidative damage. Vitamin E which is required in reproduction is a potent antioxidant found in nature. In our study, due to increasing tobacco consumption in women in especially recent years it was aimed to examine the nicotine induced damage in ovary, its mechanisms and the curative effects of vitamin E on these damages.

In our study, rats (12 week old‐ Wistar female rats) were treated with nicotine (1 mg/kg/day, intraperitoneally (ip)) and vitamin E (α‐tocopherol) (200 mg/kg/day, ip) alone or in combination in experimental groups. Levels of 17ß‐estradiol, LPO (Lipid peroxidation) and MDA (Malondialdehyde) were measured in serum of the animals. Hematoxylin and eosin (H+E) staining and labeling of Ki‐67 (a cellular marker for proliferation), 4‐HNE (4‐Hydroxynonenal; produced by lipid peroxidation in cells) and TUNEL (Terminal deoxynucleotidyl transferase (TdT)‐mediated dUTP nick end labeling;Apoptosis Detection) in paraffin sections of the ovaries was performed, immunohistochemical changes were assessed by scoring system. Ultrastructural changes in araldite sections were evaluated with the electron microscopy. The data were compared statistically.

Decreased estrogen level and increased levels of LPO and MDA in serum; decreased the number of ovarian follicles and Ki‐67‐labeling in granulosa cells of follicles; increased 4‐HNE immunoreactivity and TUNEL‐labeling were observed in nicotine‐treated rats. In these animals, nicotine co‐treatment with vitamin E significantly reduced the increased levels of LPO and MDA in serum and 4‐HNE immunoreactivity and TUNEL‐labeling in ovary sections; significantly increased serum estrogen level, the number of ovarian follicles and Ki‐67‐labeling in granulosa cells of follicles. Vitamin E demonstrated positive and curative effect on biochemical and histological parameters compared to the control group. Ultrastructurally, frequent apoptotic profiles were observed in granulosa cells in follicles in nicotine group and vitamin E co‐administration led to ultrastructural alleviation of apoptotic process.

In conclusion, our biochemical, immunohistochemical and ultrastructural level findings demonstrated that oxidative stress‐induced adverse effects caused by nicotine in folliculogenesis and streoidogenesis occur via lipid peroxidation in granulosa cells. Our data suggest that vitamin E may represent an alternative of pharmacologic treatment for preserving fertility owing to its beneficial effects on the nicotine induced damages occurred in the granulosa cells.
Z
‐stack of super‐resolved bright‐field microscopic images and its simplified reporting
Authors: Rychtarikova Renata , Stys Dalibor
Published: December 2016

We describe an algorithm of processing of z‐stacks of images of unlabelled live cells obtained from wide‐field bright‐field optical transmission microscopy to achieve the super‐resolved volumes of intracellular objects in the order of 20×20×100 nm
3
, including the classification of pixels according to an intracellular dynamics and spectral properties [1].

This super‐resolved method is based on

non‐interpolating de‐mosaicing of a 12‐bit z‐stack of raw images obtained from a camera chip equipped by a Bayer mask, which preserves as much information in the image as possible to obtain RGB images.

searching for pixels of unchanged (or near) intensities between two consecutive images, after either the simple subtraction of two consecutive images or the calculation of a point divergence gain (PDG).

The latter step of image processing is responsible for evaluation of the intracellular dynamics. If
PDG
= 0 (the same intensities at the same position in two consecutive images), large intensity homogenous non‐moving objects are mainly segmented. At minimal and maximal values of
PDG
(a pixel of the rarest intensity is replaced by a pixel of the most frequent intensity and
vice versa
), we track a large moving organelles. The other values of PDG correspond either to the other intracellular objects or to other intensities in the course of the sum of point spread functions of the live cell.

the selection and evaluation of each colour channel of the image. In the blue channel, we observe mainly autofluorescence. In the green channel, the light diffraction is also projected. In the red channel, the light absorption in near infrared region is further observed.

We also show a method of simplified reporting of resulting data on the example of a living mammalian cell.
EELS
Probing of lithium based 2‐
D
battery compounds processed by liquid phase exfoliation
Authors: Pokle Anuj , Coelho João , Macguire Eva , Downing Clive , Casey Patrick , McGuinness Cormac , Nicolosi Valeria
Published: December 2016

Two‐dimensional lithiated nanosheets usually show excellent electrochemical performance due to an increase in surface area and shorter diffusion paths. However, processing techniques, such as shear mixing or liquid phase exfoliation could induce phase changes or knock out some of the structural lithium (Li) ions, what in turn might result in poor electrochemical performance. Here different lithiated layered compounds mainly LiCoO
2
, LiMn
2
O
4
, and Li
5
Ti
4
O
12
were chemically exfoliated and investigated using electron energy loss spectroscopy (EELS) for their Li‐K edge. Further analyses were carried out, looking at the oxygen (O) K edge with their respective transition metal core loss peak (Mn, Co and Ti) which revealed changes in the Energy loss near edge structures (ELNES) when compared to the unlithiated compounds. STEM‐EELS analyses confirmed uneven distribution of lithium within the lithiated layered materials. In this work, EELS was used for the first time to detect and to probe the chemical environment of the lithium in liquid phase exfoliated material.
Understanding the Self‐Assembly of a Janus‐type
POM
‐
POSS
Co‐Cluster from low‐dose
STEM
Authors: Kuebel Christian , Yan L.T. , Wang Wei
Published: December 2016

Understanding the self‐assembly process of novel cluster‐assembled materials (CAMs) is a prerequisite to optimize their structure and function. Janus‐type molecules are of particular interest as the provide the possibility to combine dissimilar properties. In this study, two kinds of unlike organo‐functionalized inorganic clusters (polyoxometalate: POM, polyhedral oligomeric silsesquioxane: POSS) have been covalently linked by a short organic tether to form a dumbbell‐shaped Janus co‐cluster. Their supramolecular assembly in nanocrystals has been analyzed in 2D and 3D using low‐dose HAADF‐STEM techniques to understand the assembly process based on the structure and the structural variations. The results have been compared to coarse grained molecular simulations.

STEM tomography reveals a honeycomb superstructure constructed by hexagonal close‐packed cylinders of the smaller POSS cluster and an orderly arranged framework of the larger POM cluster either in single or bicrystals (Fig. 1). A more detailed analysis (Fig. 2) reveals a bilayer arrangement of the POM‐POSS clusters within the hexagonal framework as the favorable structural motive. However, locally significant structural variations are observed, both in terms of the POM neighbor coordination (Fig. 2E) as well as for the tessellation of the supercell arrangement (Fig. 2F). This self‐organization demonstrates two synergistic effects controlling the structure formation: phase separation of the two immiscible blocks favoring a hexagonal framework, while crystallization or at least ordered packing of the POM clusters favors an extended bilayer leading to a geometrically frustrated structure. Close to the growth surface, this frustration leads to large structural variations with an increasing number of clusters in the basic motive, while it becomes more defined close to the core of the nanoparticles (Fig. 2D).

A series of coarse grained molecular simulations reveals that the size asymmetry of the two clusters has a significant effect on the self‐assembled structure. Ratios between 1.5 and 1.8 lead to hexagonal structures in good agreement with the experimental results. This understanding opens up new opportunities for generating novel CAMs for advanced applications.
Investigating Surface Plasmon‐Enhanced Local Electric Fields by
EELS
with tunable <60
meV
Energy Resolution
Authors: Abellan Patricia , El‐Khoury Patrick Z. , Hage Fredrik S. , Cottom Josh , Joly Alan G. , Hess Wayne P. , Brydson Rik , Ramasse Quentin M.
Published: December 2016

Recent improvements in energy resolution, enabled by the use of electron energy monochromators, have the potential to turn EELS into a tool able to provide quantitative information of localized surface plasmons (LSPs), such as damping effects in single particles and electron kinetics of single plasmon modes.[1] Crucial to the prospect of quantitative analysis of LSPs is the requirement that the experimental energy resolution must be better than the natural line width of the plasmon resonances, all the while retaining high enough signal‐to‐noise ratio to enable an accurate determination of the properties of interest.[1] The energy resolution of EELS is customarily determined by the full‐width at half‐maximum (FWHM) of the zero‐loss (ZL) peak. The plasmon resonances, lying in the low‐loss regime, often overlap with the broad tail of the ZL peak, blurring many spectral signatures of interest. Until now, several processing techniques had to be applied to overcome these issues, relying for instance on deconvolution algorithms [2] which can introduce artifacts [3] A new generation of electron monochromators now allows for high signal‐to‐noise ratios while varying the energy resolution controllably, down to the 10meV regime [4].

Here we present recent results aimed at spatially and spectrally resolving the plasmon resonances of individual plasmonic nanostructures and of functional plasmonic devices using a Cs‐corrected and monochromated Nion UltraSTEM 100MC (‘Hermes’) microscope with a nominal energy resolution of 10meV. Figure 1 shows spatially resolved LSP modes of two individual Ag particles with different shapes and different number of crystalline domains for the energy ranges indicated in the figure. Both shape and crystallinity appear to affect the plasmonic response. We show how the energy resolution, which also affects the attainable signal‐to‐noise ratio and dictates the required integration (exposure) time, can be conveniently set and tuned, depending on the inherent properties of the system of interest. Figure 2A shows as‐recorded EEL spectra taken from the centre of a Ag nanowire (inset) and showing a narrow bulk plasmon resonance at 3.85eV. We note that the FWHM of the peak does not decrease with decreasing energy resolution from 40meV to 16meV, meaning that 40meV must be below the natural line width of the resonance. In this context, we will discuss the prospect of not only characterizing bare metallic nanostructures, but of also interrogating chemically functionalized plasmonic nanostructures using EELS. We note that the accessible energy ranges (sub‐40 meV) also allows us to probe molecules adsorbed onto metal nanostructures. In the raw spectra in Fig. 2B taken from different locations within a sample, spectroscopic signatures of aromatic thiols chemisorbed onto multiple Ag nanoparticles are shown.[5]
Study of mitochondrial networks in pancreatic islet β‐cells by
SBF
‐
SEM
Authors: Leapman Richard , Hoyne Jake , Kuo Bryan , Calco Gina , Zhang Guofeng , Aronova Maria
Published: December 2016

Mitochondria play an essential metabolic role in all eukaryotic cells because they are the organelles that provide energy to drive chemical reactions. Mitochondria are highly dynamic and change their shape between discrete structures and large interconnected networks by selective fission and fusion of their membranes. The resulting fused three‐dimensional networks can permeate through an entire cell, but their advantages, relative to isolated discrete organelles, are not fully understood. It has been suggested that networks might have the following functions: dampen biochemical fluctuations; provide a mechanism for quality control and selective mitophagy; enable diffusion of proteins; and transmit calcium signals and mitochondrial membrane depolarization throughout the cell [1, 2]. Despite their highly dynamic state, useful structural information can nevertheless be obtained from electron microscopy by studying the organization of mitochondrial networks at a given time point.

We have used a SIGMA VP (Zeiss Inc.) scanning electron microscope (SEM), equipped with a 3View (Gatan Inc.) serial block face (SBF) system [3], to quantify the connectivity of mitochondrial networks in entire insulin‐secreting
b
‐cells of mouse pancreatic islets of Langerhans. Previous work has suggested that alterations in mitochondrial fission and fusion might play a role in nutrient‐induced
b
‐cell apoptosis with possible involvement in the pathophysiology of Type 2 diabetes [4].

Previously, we found that manual segmentation of SBF‐SEM mitochondrial volumes in a single
b
‐cell took about one week for a trained operator, which precluded analysis of multiple cells within a reasonable time. We have explored a faster and more practicable approach by making use of tools within the Amira visualization software package (FEI Inc.) to partially automate the segmentation process, and enabling an entire cell to be analyzed in approximately two hours (Figure 1). Using this approach we were able to analyze quantitatively mitochondrial networks in individual pancreatic
b
‐cells in terms of the total mitochondrial volume, average volume per network, total network length, and average network length. We found that approximately one‐third of the mitochondrial volume was contained in networks of length less than 3 µm, one‐third in networks of length between 3 µm and 10 µm, and one‐third in highly fused networks of length between 10 µm and 60 µm (Figure 2).

SBF‐SEM thus provides quantitative data on the organization of mitochondrial networks, making it feasible to test computational models for mitochondrial fusion and fission.
Effect on the
SEM
topography of different sample preparation methods for thin‐film‐composite membrane
Authors: Huang Wenqing , Miao Xiaopei
Published: December 2016

Thin‐film‐composite (TFC) membrane was prepared through interfacically polymerizing m‐phenylene‐diamine (MPD) and trimesoyl chloride (TMC) on top of a poly‐sulfone substrate to form an ultrathin active polyamide layer, which is excellent in hydrophilicity, mechanical stability, thermal stability and hydrolytic stability. Scanning electron microscopy (SEM) is suitable for direct observation of TFC membrane structure, especially for superfine structure of ultrathin functional layer. The skin layer of TFC membrane is much denser than ultrafiltration membrane and its microstructure is more difficult to observe by SEM with regular way. At present, there are two common methods for fracturing membrane: brittle fracture of liquid nitrogen, embedded section. In addition, there are two less common methods with the help of precision instruments, like Focused Ion Beam cutting and Ion Milling technologies. We compare the advantages and disadvantages of four methods, and put forward a more convenient, practicable and effective method for SEM cross‐sectional analysis.

(1)Embedded Section: This method will be finished after embedding in epoxy resin, solidifying in thermostat and slicing by microtome. It has strict technical requirements for the operator. The cross section is damaged badly in the Fig.1.

(2)FIB cutting: We obtain the cross‐sectional sample using Carl Zeiss AURIGA Cross Beam FIB/SEM. FIB probe is Ga ion beam. The operating voltage and current is 30kV and 50pA respectively. The ion beam intensity is too high to keep the integrity of film structure. The cortex is damaged in the Fig.2. So this method is not suitable for soft materials.

(3)Ion milling: The process is completed by IM4000 of HITACHI. We select the section grinding mode to reduce the damage of structure, with the help of liquid nitrogen cooling mode. Although this method is somewhat better than FIB cutting, we still can't see the cortex distinctly.

(4)Brittle Fracture of Liquid Nitrogen: The cost of this method is very low. Although it's easy to use without the aid of other equipment, the SEM microstructure is difficult to observe directly and clearly. Fig.4a shows the low and high magnification images. We can see the film cross section is distorted and the cortex sagged.

The above methods have many limitations, because the toughness of membrane is too big to fracture easily. We have developed an improved sample frozen and fractured technology: the TFC membrane has swollen in ethanol at room temperature for several minutes, and then we put the sample in liquid nitrogen and fracture it with a certain slope. The functional cortex can be observed directly and effectively in the Fig.4b below.
In vivo visualizing the generation of hypochlorous acid using a novel selective fluorescent probe
Authors: Wang Haolu , Gravot Germain , Liang Xiaowen , Thorling Camilla , Zhang Run , Liu Xin , Roberts Michael
Published: December 2016

Introduction

Hypochlorous acid (HOCl) is one of the most important reactive oxygen species, and plays a vital role in physiological events and diseases. Many fluorescent probes have been synthesized to detect HOCl. However, most of them were difficult to be distinguished from endogenous fluorophores in biological samples in vivo and can only be employed under one‐photon microscopy, often with drawbacks during further application. To enhance the sensing sensitivity and reduce photodamage, we developed a novel iridium(III) complex‐based fluorescent probe for
in vivo
visualizing the generation of HOCl at the cellular level.

Methods

A novel iridium(III) complex‐based Fc (ferrocene) dual‐signaling chemosensor (Ir‐Fc) was designed and synthesized as a highly sensitive and selective fluorescent probe for the
in vivo
recognition and detection of HOCl. The absorption and fluorescent properties were investigated under simulative physiological conditions. It is well documented that during hepatic ischemia‐reperfusion (I/R) injury, HOCl is generated by neutrophils and diffuses into hepatocytes, causing oxidant stress‐mediated injury. Therefore, we used a BALB/c mouse hepatic I/R injury model to test this probe. DermaInspect system (JenLab GmbH, Jena, Germany) was used for
in vivo
multiphoton microscopy (MPM) imaging of the liver after injection of 0.2 mL Ir‐Fc (50μM) from the portal vein. Systemic toxic effects were evaluated by examining the pathological changes of the major organs of mice.

Results

Figure 1A shows the reaction mechanism of the fluorescent response of Ir‐Fc toward HOCl. Ir‐Fc exhibited a strong positive fluorescent response only in the presence of HOCl, whereas negligible fluorescent signals were observed upon the additions of other reactive oxygen/nitrogen species and metal ions (Figure 1B). The dose‐dependent fluorescent enhancement followed a good linear relationship with HOCl concentration (Figure 2). In mouse liver with I/R injury, reduced autofluorescence was detected by MPM, indicating the hepatocyte necrosis (Figure 3D). As shown in Figure 3F, remarkable enhancement of the red fluorescence was observed in hepatocytes with decreased autofluorescence, indicating the reaction of Ir‐Fc with endogenous HOCl molecules. No obvious toxic effects were observed in histological examination of major organs after Ir‐Fc injection.

Conclusion

Ir‐Fc has good biocompatibility, low cytotoxicity, high specificity to HOCl, and rapid “off‐on” fluorescence. This complex is suitable as an
in vivo
HOCl detection probeat the cellular level.
Polar‐graded multiferroic
S
r
M
n
O
3 thin films
Authors: Guzman Roger , Maurel Laura , Langenberg Eric , Lupini Andrew R. , Algarabel Pedro A. , Pardo José A. , Magén César
Published: December 2016

Engineering defects and strains in oxides provides a promising route for the quest of thin film materials with coexisting ferroic orders, multiferroics, with efficient magnetoelectric coupling at room temperature. Indeed, precise control of strain gradients would enable custom tailoring of multiferroic properties, but presently remains challenging. Here we use aberration‐corrected scanning transmission electron microscopy (STEM) to explore the existence of a polar‐graded state in epitaxially‐strained antiferromagnetic SrMnO
3
thin films, whose polar nature was predicted theoretically [1] and recently demonstrated experimentally [2]. Combining annular bright field (ABF) with high angle annular dark field (HAADF) imaging we map at the atomic scale level the cation‐oxygen dipole distortion far from (Figure 1) and near to the domain walls (Figure 2). Local deformation analyses reveal a particular strain state where flexoelectricity –the coupling between strain gradients and polarization– induce a rotation of the in‐plane 110 electric polarization due to 001 strain gradients. The analysis of the local Mn oxidation state by electron energy loss spectroscopy (EELS) reveals that the strain gradients are related to a gradual distribution of oxygen vacancies across the film thickness. Here we present a chemistry‐mediated route to induce flexoelectric polar rotations in oxygen‐deficient multiferroic films with potentially enhanced piezoelectricity.
Investigation of plasticity/fatigue mechanisms at interfaces in
N
i using ex‐situ and in‐situ
SEM
/
TEM
micro/nano‐mechanical testing
Authors: Samaeeaghmiyoni Vahid , Groten Jonas , Idrissi Hosni , Schwaiger Ruth , Schryvers Dominique
Published: December 2016

The present work focuses on the fundamental plasticity/fatigue mechanisms operating at interfaces in micro/nano scale Ni samples.
In‐situ
SEM fatigue tests have been performed on FIB prepared single and bi‐crystal micropillars with well‐known orientations as revealed by EBSD. Careful characterizations of the nature and the distribution of deformation dislocations, the character and the local structure of the interface as well as the mechanisms controlling the interaction between these defects under cyclic loads were performed using
ex‐situ
TEM techniques including diffraction contrast imaging, automated crystallographic orientation and nanostrain mapping in TEM (ACOM‐TEM) as well as electron tomography.

The primary TEM results obtained on single crystal micropillars after fatigue tests revealed the presence of dislocation walls structure as shown in Figure 1(a) and (b). ACOM‐TEM revealed local changes of crystal orientation around 1–2 degrees at the position of the dislocation walls. Furthermore, systematic contrast analysis of dislocations in these areas confirmed that only slip systems with the highest Schmid factor have been activated. The analysis of micropillars with GBs subjected to fatigue tests has shown the accumulation of dislocations at the GBs (Figure 1(c) and (d)) without slip transfer or localized plasticity. Electron tomography has been used to investigate the 3D distribution and the interaction of deformation dislocations within the dislocation walls.

In order to directly observe the plasticity mechanisms, quantified
in‐situ
TEM tensile tests were performed on both single and bi‐crystal samples using the Pi 95 picoIndenter instrument and a MEMS device called ‘’Push‐to‐Pull’’ (PTP) from Hysitron.Inc (Fig 2). In order to minimize the effect of FIB on the
in‐situ
tensile samples, an original sample preparation method combining twin jet electro‐polishing and FIB was used, see figure (2). Nucleation‐controlled‐plasticity has been observed with defects induced by FIB at the edges of the sample acting as preferential sources for the nucleation of deformation dislocations. Furthermore, the in‐situ TEM nanotensile experiments revealed the elementary mechanisms controlling the interaction between dislocations and pre‐selected GBs from the electro‐polished thin foils, as the nucleation of dislocations from GB, see figure (3). These results indirectly shed light on the micropillar's behaviour as the root cause of the deformation is connected to the FIB preparation and the dislocation/GB interaction mechanisms.
Nanoscale Chemical Mapping of
G
dX3@
WS
2 Nanotubes by
EDS
‐
STEM
Tomography
Authors: Anumol E. A. , Deepak Francis Leonard
Published: December 2016

The hollow interiors of nanotubes could host the growth or filling of foreign elements/compounds to obtain hetero‐structures. The growth of these materials in the confined one dimensional space lead to novel properties. Capillary filling serves as a method to enable filling of carbon nanotubes and inorganic nanotubes including those of BN and WS
2
.
1, 2
In this work, considering the biocompatibility of WS
2
and paramagnetic property of gadolinium (III) compounds, capillary filling is employed to obtain GdX
3
@WS
2
nanotubes (X=Cl, Br, I). The precise determination of the structure and composition is detrimental in its further application. Thus in the present study the morphology, structure and chemical composition of the synthesized GdI
3
filled WS
2
is investigated using aberration corrected scanning/transmission electron microscopy and associated spectroscopic techniques (EELS and EDS). The three‐dimensional morphology is investigated using HAADF‐STEM tomography but obtaining three dimensional composition information is non‐trivial due to the presence of multiple high atomic number elements. Therefore, EDS‐STEM tomography is employed in the present study to map the chemical composition in three dimensions.
3
In order to reduce the beam induced effects on the specimen, tomography experiments were carried out at 80 kV in the present case. In view of the long duration of electron beam exposure necessary to perform EDS‐STEM tomography, additional electron irradiation studies were carried out to optimize the EDS‐STEM tomography conditions.
Surface plasmon coupling revisited with electron energy loss spectroscopy
Authors: Das Pabitra , Martins Hugo Lourenço , Tizei Luiz , Kociak Mathieu
Published: December 2016

In the last fifteen years or so, a significant amount of research activities took place in the field of metal nanoparticle plasmonics probed by fast electron beam. Local electron probe techniques like electron energy‐loss spectroscopy (EELS) and cathodoluminescence (CL) have advanced very fast during this time. Both these techniques helped us to gain considerable insight into the plasmonic properties of metallic nanostructures [1,2]. One of the many interesting nanostructures are the metal nanoparticle dimers. They can confine a huge amount of field in the gap. As the modes can be tuned precisely by changing the separation between the nanoparticles, they bear the promise to be used as sensors. It is well known that as the two individual particles approach each other, bonding and antibonding modes appear. Bonding and antibonding corresponds to in‐phase and out‐of‐phase interaction of the individual particle plasmons. Analogous to the molecular orbital theory, the bonding mode appears at lower energy and the antibonding mode at higher energy with respect to the individual particle plasmon mode [3]. With electron beam we can probe the precise location of the particle dimer to see which mode is excited at what location. Even though there is a wealth of literature on the plasmonic modes of individual metallic nanostructure, a systematic experimental study of coupled plasmons is deficient.

The central idea of the current work is to develop a deeper understanding on the coupling of the nanoparticle plasmons when they are brought close to each other (a few nanometers). For this purpose, we have chosen a cross shaped metal nanoparticle (Figure 1). We choose this structure for easy tuning of plasmon modes along the length of the rods by changing the rod length and because it is easy to model numerically. To have a detailed idea on the underlying physics, we start with the basic building block of the cross, i.e. a single nano rod. Then we increase the complexity of the structure by adding two rods perpendicularly to make a cross, and finally bringing two crosses close together to make a dimer. We make EELS on all of these structures and see the evolution of the modes. In this way, we will be able to explore the exact formation of different modes and the coupling between them.

To realize our idea, we have performed electron beam lithography to make silver nanostructures on Silicon Nitride substrate. EELS experiments are performed using a scanning transmission electron microscope (STEM) fitted with homemade EELS detection system [4]. To gain insight about the plasmonic modes, we perform 3D boundary element method (BEM) simulations [5] and compare with the experimental data. A representative EELS spectrum and the 2D plasmon maps has been shown in Figure 2.

In the conference, we will present and discuss our experimental and simulation results. This will provide new insight into the physics of plasmon coupling.
Inversion of dynamical scattering from large‐angle rocking‐beam electron diffraction patterns
Authors: Wang Feng , Pennington Robert , Koch Christoph
Published: December 2016

A method for ab‐initio structure factor retrieval from large‐angle rocking‐beam electron diffraction (LARBED)[1] data of thin crystals is described and tested.

This method determines crystal structure factors and specimen thickness from the intensities of the diffraction spots alone, solving a nonlinear least‐squares problem. No additional information, such as atomicity or information about chemical composition, have been made use of.

In addition to a demonstration of the method on 120 keV experimental data of SrTiO
3
, where 456 structure factors have been retrieved, we analyze the dependence of the success of the reconstruction on specimen thickness and tilt range by applying this algorithm to simulated data. Our numerical experiments show that the dynamical inversion by gradient optimization works best if the beam tilt range is large and the specimen not too thick. At specimen thicknesses which allow for moderate multiple scattering, the large tilt amplitude effectively removes local minima in this global optimization problem, making ab‐initio structure factor retrieval possible.

In addition, a dynamic parallelism framework  based on the Compute Unified Device Architecture (CUDA) is introduced, reducing the runtime of the optimization routine by two orders of magnitude when compared to running on a CPU.

The authors acknowledge funding by the Carl Zeiss Foundation as well as the German Research Foundation (Grant no. KO 2911/7‐1 and SFB 951).
Dislocation Modelling: Calculating
EELS
Spectra for Edge Dislocation in Bismuth Ferrite
Authors: Agrawal Piyush , Rossell Marta D. , Hébert Cécile , Passerone Daniele , Erni Rolf
Published: December 2016

Recent advances in electron energy‐loss spectroscopy (EELS) triggered by the implementation of aberration correctors and novel spectrometers have enabled atomic resolution and single atom sensitivity. The energy‐loss near‐edge structure (ELNES) in core‐loss EELS provides insight into the electronic structure of individual atomic species containing information about their bonding characteristics such as, e.g., oxidation state, charge transfer and site coordination. Yet the electronic structure information is buried in the spectral fine structure which can be regarded as a “fingerprint” of the atom's bonding characteristics. In order to overcome this shortcoming of a purely experimental approach, the ELNES of core‐loss EELS ionization edges can be obtained from first‐principles electronic structure calculations.

BiFeO
3
(BFO) is a multiferroic perovskite that exhibits antiferromagnetism coupled with ferroelectric order. Besides, because of their astounding electromechanical properties, BFO thin films are promising candidates for the replacement of lead‐based ceramics in microelectromechanical system devices. It is well known that performance of ferroelectric devices is reduced by the presence of crystal defects such as edge dislocations. This type of crystal defects within this material
1
are due to the lattice mismatch between the BFO film and the SrRuO
3
substrate as strain compensation mechanism. However, the electronic structure close to the dislocation core is not yet well understood. In this work, we investigate the influence of edge dislocations on the material's local electronic properties, using a combined experimental and theoretical strategy based on HAADF‐STEM, EELS and atomistic simulations.

In this study the edge dislocation within BFO was modelled based on Peierls‐Nabarro Model (P‐N model)
2
theory. The initial guess was obtained from the P‐N model and further optimized using BVVS classical potentials for BFO
3
as incorporated in the LAMMPS package
4
. Fig 1 shows the final geometry of the edge dislocation model obtained after optimizing it with this potential. FEFF9
5
, a real space multi‐scattering Core level spectroscopies code will be used on the final optimized model to obtain the O K‐edge and Fe L‐edge spectra at the dislocation core. Fig 2 shows the experimentally recorded O K‐edge EELS on a line of atomic columns (points 1 to 9) crossing the dislocation core. The comparison of the experimental spectrum with the calculated EELS allows shedding atomistic insight on the EELS peak structure. In particular, it is possible to explain to which extent a defect in a bulk material (in this case, an edge dislocation) locally affects its electronic properties, thus enhancing the power of electron energy‐loss spectroscopy in the high‐resolution description of complex materials.
Preparation of high fidelity holographic vortex masks using advanced
FIB
milling strategies
Authors: Schachinger Thomas , Steiger‐Thirsfeld Andreas , Löffler Stefan , Stöger‐Pollach Michael , Schneider Sebastian , Pohl Darius , Rellinghaus Bernd , Schattschneider Peter
Published: December 2016

Holographic masks (HMs) with dislocation gratings placed in the condenser system of a TEM have been proven to be a reliable and robust method to impart quantized orbital angular momentum (OAM), as well as quantized magnetic moment onto the imaging electrons [1]. These so‐called electron vortex beams (EVBs) gathered a lot of attention due to some unusual properties like topological protection [2], peculiar rotation dynamics in magnetic fields [3] and intrinsic chirality. It has been suggested to use a holographic vortex mask as a vorticity filter after the specimen, in the selected area aperture holder, in order to detect spin polarized or other chiral transitions. This would bring up the unique chance to study magnetic properties of amorphous or nanocrystalline materials because the specimen's role as a crystal beam splitter – necessary in the standard energy‐loss magnetic chiral dichroism (EMCD) geometry ‐ is obsolete in this setup.

High fidelity HMs are needed for such experiments. Also, in order to achieve high vortex order separation, the grating periodicity should be very fine. To improve the signal‐to‐noise ratio of the EMCD measurements, the dimensions of the HMs need to be large. FIB milling proved to be a robust and reliable technique to produce HMs, but with ever‐increasing demands on structure size and fidelity, the ordinary milling strategy using raster‐ or serpentine scanning showed limited success. Therefore, we developed a new threefold milling strategy. The first step is to employ a so called “vector scan” technique, where “stream”‐files provide the possibility to fully control the position and dwell time of the ion beam to generate spiral milling paths for every hole in the HM structure (see Fig. 1). The next step is to reverse the milling order and ‐direction after each pass [4]. Inspired by [5], the last part consists of a position‐dependent dwell time reduction in the proximity of the hole edges to enhance the HM bar edge fidelity. Fig. 2 shows an exemplary 30 µm vortex mask with a grating periodicity of 500 nm and a thickness of roughly 700 nm.

One challenge encountered with this new strategy is limited digital‐to‐analog‐converter resolution as well as memory issues for large “stream”‐files. Using a state of the art FIB, it was possible to cut 50 µm HMs and to compare the ordinary raster scanning technique to the one proposed here, see Figs. 3 and 4. These results indicate that our new threefold scanning ansatz enhances the edge quality. Howerver, issues like sample‐ and beam drift as well as the crystallinity of the mask material have to be addressed in order to further improve the fidelity of the HMs' edges.