bluuebunny/crossref_metadata_2025_split huggingface dataset:
Switching mechanism of
DNA
replication fork complex revealed by single particle analysis
Authors: Mayanagi Kouta , Ishino Sonoko , Takafuji Mika , Mitsuoka Kaoru , Shirai Tsuyoshi , Kiyonari Shinichi , Nishida Hirokazu , Kohda Daisuke , Morikawa Kosuke , Ishino Yoshizumi 
Published: December 2016

A number of proteins are involved in DNA replication, which is essential for the inheritance of genetic information. These proteins assemble to form a huge complex, called replisome, and accomplish each function through highly regulated manner. Electron microscopic single particle analysis is one of the most powerful methods to study such complex system, which is difficult to study by X‐ray crystallography.
DNA replication in archaea and eukaryotes is executed by family B DNA polymerases, which exhibit full activity when complexed with the DNA clamp, proliferating cell nuclear antigen (PCNA). PCNA has a trimeric ring structure that encircles the DNA, and increases the processivity of the bound DNA polymerase by tethering it to the DNA. It is known now, that PCNA also interacts with multiple partners to control DNA replication, DNA repair, and cell cycle progression, and works not only as the platform, but also as the conductor for the recruitment and release of these factors. However, the molecular architectures as well as the mechanism of the regulation of these replication factors are not known in detail.
We have been focusing our interest on the mechanism of synthesis and maturation of Okazaki fragments during lagging strand DNA replication in which three replication factors, i.e. DNA polymerase, Flap endonuclease, DNA ligase, are playing essential roles (Fig. 1A‐C). As all of these 3 enzymes are known to interact with PCNA trimer, a switching mechanism between these factors has been proposed, called PCNA tool belt model, which is considered to increase the efficiency of these sequential reactions (Fig 1D). Recent biochemical study, on the other hand, suggests a sequential switching mechanism of these factors. Little is known regarding the switching mechanism, due to the lack of the structural data of these complexes.
We have investigated the three‐dimensional structure of the core components of the replisome, such as DNA polymerase B(PolB)‐PCNA‐DNA, and DNA ligase‐PCNA‐DNA ternary complexes, by single particle analysis (2‐ 3). Besides the authentic interaction through a PCNA‐interacting protein box (PIP‐box), we could find a novel contact between both polymerase‐PCNA and ligase‐PCNA. Mutant analysis showed that these contacts are involved in the regulation of the replication factors, such as the switching between the polymerizing and editing modes of the PolB. Our results, showing that both factors interacting with two subunits of the PCNA trimer ring, were inconsistent with the standard tool belt model. However, the third PCNA subunit was still free in both complexes, thus we analyzed the complex structures with two replication factors bound to a PCNA ring, in order to investigate the switching mechanism between them in more detail.
Combining
TEM
and
APT
for a better understanding of super high efficiency Cu(In,Ga)Se2 thin film solar cells
Authors: Castro Celia , Raghuwanshi Mohit , Pareige Philippe , Duguay Sébastien , Cadel Emmanuel , Witte Wolfram , Jackson Philip , Hariskos Dimitrios , Kessler Friedrich , Buecheler Stephan , Carron Romain , Avancini Enrico , Bissig Benjamin , Tiwari Ayodhya 
Published: December 2016

The Sharc25 EU H2020 project aim is to push the efficiency of Cu(In,Ga)Se2 (CIGS) thin film solar cells to their maximum theoretical limit. Sharc25 consists in a large consortium to take under consideration both theoretical and experimental aspects. One of the strategies to achieve this goal is to improve and understand the CIGS absorber material deposited by co‐evaporation.
The polycrystallinity of the CIGS absorber layer is one of the key elements which plays a crucial role in its efficiency. We combine here atom probe tomography (APT) (Fig. 2) and transmission electron microscopy (TEM) to study the distribution of alkali elements at sub‐nanometer scale and with statistic relevancy. 3D APT atomic mapping of grain boundaries (GB) is here completed by systematic STEM‐EDS analyses of GB all along the CIGS absorber layer (Fig. 1). Issues on samples preparation for APT and TEM will be also discussed.

This project has received funding from the
European Union's Horizon 2020 research and innovation programme
under grant agreement No 641004
Im2Cr
: An efficient tool for crystallographic indexing of
HR(S)TEM
images
Authors: Silva André , Carbó‐Argibay Enrique , Proença Alberto , Stroppa Daniel 
Published: December 2016

Transmission Electron Microscopy (TEM) and Scanning TEM (STEM) have been widely used to characterize nanostructured materials with atomic resolution, and significant advances on their experimental setup greatly extended the current pool of analysis possibilities at the nanoscale. The exploration of advanced (S)TEM characterization capabilities and their reproducible application to reach a suitable sampling is often restricted by the extensive data analysis procedures required to reliably interpret experimental results and to extract quantitative information. Even routine tasks such as nanoparticles crystallographic indexing from electron diffraction patterns or from high resolution (S)TEM images are mostly carried out manually by the users, resulting in a reduced TEM characterization yield and significant user bias.
This work presents Im2Cr, a new software tool to aid the crystallographic indexing of nanostructured materials using high resolution (S)TEM images. Im2Cr implementation aims for a minimal user interaction, supporting the detection of zone‐axis oriented particles, and including an efficient peak detection process applied to the images Fourier Transform (FT). With basis on the FT peaks distances and relative angles, crystallographic indexation is carried out autonomously via comparison with a list of candidate structures named by the user, and a ranking of the best matching combinations of crystallographic structures and viewing zone axes is generated.
Im2Cr was successfully tested for robustness and execution efficiency in a wide range of High Resolution (S)TEM images from crystalline nanomaterials, with domain size ranging from 4 to 100 nm. The autonomous indexation with preset parameters has a very high success rate, and runs in a small fraction of typical (S)TEM images acquisition time by taking advantage of the inherent hardware parallelism. Alternatively, the user can operate Im2Cr in a semi‐autonomous mode and control relevant parameters related to the region of interest (ROI) selection on the (S)TEM image and on the FT peaks detection. Im2Cr promising results point to the possibility of real‐time image analysis with reduced user interaction, allowing for an increased (S)TEM characterization yield and also enabling the interpretation of complex images, such as those from nanocrystalline materials imaged in high‐order zone axis orientations.
Epitaxy of
G
a
N
nanowires on graphene
Authors: Kumaresan Vishnuvarthan , Largeau Ludovic , Madouri Ali , Glas Frank , Zhang Hezhi , Oehler Fabrice , Cavanna Antonella , Babichev Andrey , Gogneau Noelle , Tchernycheva Maria , Harmand Jean‐Christophe 
Published: December 2016

Nitride nanowires (NWs) are today actively explored as an active material for a large number of optoelectronic devices (light emitting diodes, photodetectors, solar cells). The NWs are usually grown on bulk crystalline substrates (Si, sapphire) but these substrates impose their properties which may not be adapted to the device functionality (low electrical or thermal conductivity, opacity, weigh, rigidity, cost,…). Recently, graphene has been proposed as an attractive candidate to grow III‐V semiconductor NWs [1]. Graphene is transparent, flexible and it has high thermal and electrical conductances and it can be synthesized at low cost on large areés. Furthermore, graphene films are easily transferable to almost any carrier substrate, including amorphous and/or flexible materials.
In this study, epitaxial growth of GaN nanowires on graphene is demonstrated using molecular beam epitaxy without any catalyst or intermediate layer. We have grown GaN NWs on isolated mono‐crystalline graphene flakes transfered onto SiO2/Si carrier substrates. The nanowires grow vertically and X‐Ray diffraction (XRD) show that the nanowires have grown along their c‐axis. We have observed by SEM that locally, the NW factes have the same in‐plane orientation over several µm². The 10‐10 facets of the NWs have been determined by high angle annular dark field scanning transmission electron microscopy (HAADF‐STEM). Cross‐sections have been prepared by focussed ion‐beam (FIB). HAADF images of NW‐graphene/silica interface show that the base of the NWs is defect‐free. Selected area electron diffraction (SAED) patterns taken from different zones show identical patterns indicating that the nanowires from all the zones have same in‐plane orientation. Since it is well established that the hexagonal periphery of the graphene flakes corresponds to zig‐zag edges of the graphene lattice, we can conclude that the epitaxial relationship is 11‐20 GaN parallel to the graphene zig‐zag. It leads to an apparent mismatch of 29% between the graphene and the GaN wurtzite structure. This value is very large. We show that an in‐plane super cell coincidence between 4 unit cells of graphene and 3 unit cells could lead to a more favorable mismatch of ‐3.1%. The optical properties of NWs arrays have been probed by photoluminescence spectroscopy.
Aberration corrected
CVD‐TEM
for in‐situ growth of
III–V
semiconductors
Authors: Wallenberg Reine , Jacobsson Daniel , Thelander Kimberly Dick , Gustafsson Joacim , Dogel Stas 
Published: December 2016

We will show the first results from a newly designed Environmental TEM with cold FEG emitter, aberration corrector, heating stage and a free choice of up to nine different gaseous reactants to produce heterostructures in‐situ during real‐time observation and analysis.
Observing growth of semiconductors on an atomic scale, under MOCVD or CVD‐like conditions, will bring valuable information and understanding of the possibilities and limitations for making devices, solar cells and LED:s from nanowires. We have constructed a 300 kV Environmental TEM, with cold FEG emitter, heating stage and a free choice of up to nine different gaseous reactants to produce heterostructures in‐situ.
The use of an aplanatic B‐COR image corrector provides space in the objective lens polepiece for a heating stage reactor with gas inlets, while still achieving an 86 pm point resolution (in vacuum). The gas inlets are designed to give a total pressure of up to10 Pa, which is sufficient to grow nanowires at a reasonable speed. The gas handling system will allow switching between the different gases at will, or through programmed sequences.
Changes and defects in the crystal structure, and the effect on the outer shape of the nanowires can be followed by combinations of conventional HREM imaging, STEM‐DF and STEM‐BF, as well as SE‐imaging. Simultaneous analysis by XEDS will provide chemical changes along the growth path.
Temporally and spatially resolved local strain tracking microscopy
Authors: Bayraktar Halil , Aydin Onur , Aksoy Bekir , Akalin Ozge Begum , Alaca B. Erdem 
Published: December 2016

Abstract
Cell‐stretching is a key method to regulate deformation magnitude, cyclic strain levels, and frequencies, therefore elucidating the biological processes involved in activation of mechanosensitive pathways, cell patterning and morphological changes at physiologically relevant mechanical loads. Although several approaches and methods such as uniaxial or biaxial devices have been demonstrated to deform cells and usually compute ration cross‐head displacement to original length of sample as an indicator of percentage stretch, however in‐plane strain components may have a non‐uniform spatial distribution due to heterogeneous extracellular matrix of connective tissue around cells. Therefore, the average value of the applied cross‐head strain is expected to be different than local strain in the vicinity of cells. This limitation has prompted us to develop an alternative approaches.
Here, we present a novel uniaxial cell‐stretching device integrated into inverted fluorescence microscope that provides a high spatial resolution to determine the local strain changes around fluorescent and non‐fluorescent cells (RSI, 2016, 87, 023905). Transparent and biocompatible polydimethylsiloxane PDMS elastomer modified with small fluorescent beads are used to deliver uniform strain at the physiologically relevant magnitude and cycles. The design of our device for acquisition of real‐time spatiotemporal data and single‐particle tracking methods to determine bead positions that was used for computation of strain fields at various sample geometries will be described (Fig. 1). Briefly, trajectory of beads is computed by comparing each registered location in consecutive frames and minimizing the square displacement of centroids. Displacement vector is obtained from displacements and later used to calculate longitudinal normal, traverse normal and shear components of strain with relevant deformation tensor (Fig. 2). Lastly, we will discuss that HeLa S3 cells adhered to biocompatible and flexible collagen coated surface are stretched to determine simultaneously detection of morphological changes and local strains around the cells by tracking embedded fluorescent beads (Fig. 3). The method enables to measure local strain field and image adherent cells simultaneously, therefore provides accurate and time‐resolved correlation between applied mechanical deformation and cell response.
Cryo‐EMStructural characterization of the M. tuberculosisESX‐1 secreted virulence factorEspB
Authors: Tria Giancarlo , Siroy Axel , Delei Chen , Iakobachvili Nino , Furusho Hirotoshi , López‐Iglesias Carmen , Ravelli Raimond B. G. , Peters Peter J. 
Published: December 2016

Mycobacterium tuberculosis(Mtb) is the top infectious disease killer worldwide. The threat posed by this bacterium is higher than ever, with the recent discovery of multi and extended‐drug resistant (MDR, XDR) strains with improved virulence.Virulent mycobacteria infect their host cells through a Trojan Horse strategy: they get ingested by the normal phagocytosis route but instead of being digested through acidification and maturation of the phagosome to phagolysosome, these bacteria enter the host's cell cytosol where they replicate freely. Controlled death of the host cell will allow the spreading of the descendants in the infected organism.A key step in the infectious life cycle of the virulent mycobacteria is their escape from the phagosome compartment [1]. This phenomenon is mediated by the release of different proteins (EsxA, EsxB, EspB) with cytolytic activities, that will perforate the phagosome membrane. These factors are secreted by the specialized machinery ESX‐1 of the recently discovered Type 7 Secretion System family [2].The ESX‐1 machinery is a multi‐protein complex thought to span the entire mycobacterial cell envelope. It is constituted by a core of structural proteins (EccACabBDE1) and the assembly and/or functioning of the machinery seems to require the recruitment of additional proteins, including substrates.Among the different ESX‐1 substrates, the 48 kDa protein EspB seems to carry multiple roles: this secreted protein carries cytolytic activity on its own; it is co‐secreted with EsxA (the main virulence factor) and this secretion is mutually dependent; EspB may also play a structural role suggested by its ability to oligomerize as well as its proteolytic maturation by MycP1during the translocation process.Recently, the structure of the N‐terminus of EspB was published [3] showing the N‐terminal be composed of a bundle of α‐helices, similar to other ESX substrates; the C‐terminal is predicted to be unfolded. A heptameric structure was modelled computationally based on the monomeric X‐ray data in combination with negative‐stain electron microscopy 2D classes.Here, we report structural characterization of EspB using transmission electron microscopy data. We determined the structure of the N‐terminal helix bundle, the mature form as well as the pre‐protein EspB at sub‐nanometer resolution. Moreover, we were able to identify several discrete oligomeric states of the proteins that were not previously observed: these data will aid to a better understanding of ESX‐specific substrates.
Low energy electron beam induced cleaning of graphene layers in
SEM
s
Authors: Mikmekova Eliska , Frank Ludek , Mullerova Ilona , Sluyterman Seyno 
Published: December 2016

In order to examine mutually overlapped flakes of two‐dimensional crystals such as graphene with an electron microscope we need to obtain a contrast contribution from a single layer of carbon atoms. This task requires increasing the scattering rate of incident electrons by means of a drastic lowering of their energy to hundreds of eV or less.
The effect of airborne contaminants, mainly hydrocarbons, on graphene layers distinguishing in low energy S(T)EMs can be significant and due to their removing from the surface by appropriate in‐situ methods is very important from practical point of view. During the scanning of the surfaces by electrons, the image usually gradually darkens because the hydrocarbon layer is deposited on the top. This effect can be described as an electron stimulated deposition, the thermal diffusion of organic molecules around the irradiated area is use as a source of building atoms, precursor. On the other hand, the effect of electron stimulated desorption occurred at the same time, especially at low observation energies, and then depends which process, deposition or desorption, is dominated. Our experiments have showed the fact that prolonged bombardment with electrons in a range of hundreds or even tens eV gradually increases the transmissivity (and decreases the reflectivity) of graphene due to the removal of adsorbed gas molecules providing an ultimate cleaning procedure evidently leading to an atomically clean surface. A sample such as graphene enables one to distinguish this kind of cleaning from radial damage, so this result opens up new possibilities for certain surface studies performed without ultrahigh vacuum.

Experiments have been performed in a FEI SEM/STEM microscope equipped by beam deceleration mode on free standing graphene (Ted Pella
®
) and the effects of landing electron energy, samples biasing, electron dose, heating, etc. on the cleaning efficiency of electrons was studied in details. Moreover, the structural damage of the graphene after extremely high electron doses was observed, and the critical values for selected impacted energies were also calculated.
10 bar nanoreactors for in situ transmission electron microscopy
Authors: Pandraud Gregory , Morana Bruno , Wei Jia , Juffermans Casper 
Published: December 2016

Environmental transmission electron microscopy (ETEM) is becoming an increasingly important field of study as it is possible to investigate the material‐environment interactions on a nanoscale, the scale at which most of these interactions initiate. In a TEM, this can be achieved by one of the following approaches: the opened type, using a differentially pumped vacuum system where the reactive gases are spread around the specimen area of the TEM; and the closed type, using a windowed environmental cell. In the first case the maximum achievable pressure around 1 bar [1] but it is limited to flat samples and for the second type (where two chips are often put on top of each to obtain a closed cell) the maximum pressure rarely exceeds 4 bars [2]. However the last configuration is much closer to what happens in industrial applications.
We present in this paper, a new MEMS nanoreactor fully integrated on a single die. It enables atomic‐scale imaging of nanostructured materials under the high pressures and temperatures that are typical for many industrial applications (10 bar and 650°C). The reactor can therefore be used to study the behavior of e.g. catalysts in a transmission electron microscope (TEM). It has a channel of 5 µm (allowing therefore an efficient loading of the samples to study), which is made with surface micromachining techniques and contains pillars that prevent bulging. The channel high can be adjusted by adjusting the right layers in the devices processing. Integrated with the device are 22 very thin windows (20 nm) and a resistive heater. The material chosen for the heater is Molybdenum. It offers a very high stability at 650 C for up to 10 h. The reactor is very transparent and clean (the windows being etched in a such way that contamination/residues are prevented) enabling the imaging of atomic lattice fringes with a spacing down to at least 0.15 nm. The maximum working pressure measured over 50 devices is above 9 bars in 90% of the cases.
Super‐resolution fluorescence microscopy of cryo‐immobilized samples
Authors: Kaufmann Rainer , Hagen Christoph , Grünewald Kay 
Published: December 2016

Correlative light and electron microscopy (CLEM) benefits greatly from the development of super‐resolution fluorescence microscopy. With a resolution down to the 10 nm range it enables to bridge the large resolution gap between the two different microscopy techniques. However, equally important as the achievable resolution of the imaging system is the preservation of the biological structures during the sample preparation process. Imaging undisturbed structures in living cells remains very challenging for super‐resolution fluorescence microscopy with its relatively long acquisition times. Typically, chemical fixation is used to immobilize the sample for achieving best technical results, but unfortunately this is associated with structural changes in the sample, especially at a level below the diffraction limit of light [1,2].
Cryo‐immobilization offers a preferable alternative. Here, fast freezing techniques enable vitrification of the sample and preserve the structures in a near‐native state. Although cryo‐immobilization has been established as a routine technique in the fields of electron and X‐ray microscopy, it was long unclear whether super‐resolution fluorescence microscopy could be performed under cryo‐conditions to image vitrified samples [3].
Recently, we demonstrated on the single molecule level that photo‐switching of fluorescent proteins is possible under cryo‐conditions and suitable for the super‐resolution method of single molecule localization microscopy (SMLM) [4]. Chang et al. and Liu et al. have shown the correlation of cryo‐SMLM with electron cryo‐microscopy [5,6]. Here, we present cryo‐SMLM of vitrified biological samples and its prospects for cryo‐CLEM. We demonstrate that a resolution improvement of up to 5x compared to conventional fluorescence cryo‐microscopy is possible.
Super‐resolution fluorescence cryo‐microscopy offers the possibility to image fluorescently labelled biological samples with diffraction‐unlimited resolution, immobilized, but with structural preservation in a near‐native state. This is not only helping to bridge the large resolution gap in cryo‐CLEM, but might also offer an alternative to the dilemma in conventional super‐resolution imaging, where one has to choose between the limited temporal resolution in case of living cells or the disadvantages of chemical fixation.