bluuebunny/crossref_metadata_2025_split huggingface dataset:
Deconvolution of
EDS
steel spectra using low acceleration voltages and low energy
X
‐ray lines
Authors: Terborg Ralf , Salge Tobias , Pinard Philippe , Richter Silvia 
Published: December 2016

One of the most important materials used in industry is steel. Its fine microstructure consisting of different phases and inclusions, has led to the development of new steel alloys that push the analytical requirements for spatial resolutions down to the 100 nm scale. In X‐ray microanalysis either using energy or wavelength dispersive spectrometry (EDS, WDS), low accelerating voltages, e.g. ≤ 6 kV, can fulfill this requirement. At low accelerating voltages only the L lines for most steel alloying elements (e.g. Cr, Mn, Fe, Co, Ni) can be determined as their K lines can no longer be excited. Qualitative and quantitative analysis of low energy L lines is very challenging due to the presence of absorption edges within the bremsstrahlung background, the energy dependence of the efficiency and the uncertainties of absorption effects [1]. Peak overlaps, furthermore are considerable for EDS and can even be significant for WDS.
Due to these effects this type of analysis poses difficulties for EDS and WDS. To evaluate and compare these challenges for both techniques, a systematic study was performed on steels at acceleration voltages of 15 kV and 5‐6 kV [1,2]. Spectra and net intensities of 15 steel samples covering a wide range of concentrations of the major elements were simultaneously acquired on a JEOL JXA8530F electron microprobe equipped with 5 WD spectrometers and a Bruker XFlash ED spectrometer.
The results of the 15 kV spectra show that for elements with a concentration of < 1 wt.% the WDS results are more accurate when significant peak overlaps occur in ED spectra (Mn, Co, Cu). For elements without peak overlaps, EDS results were comparable (Si, Mo) to WDS [2]. At an acceleration voltage of 6 kV, the deconvolution results of ED spectra show agreement with the experimental spectra, fig. 1. Since the quantification of L lines is more sensitive to the quantification procedure and fundamental parameters (e.g. mass absorption coefficient) used, the comparison focuses on k‐ratios instead of the quantified mass fractions. Fig. 2 shows a good agreement between EDS and WDS k‐ratios, substantiating the peak deconvolution procedure shown in fig. 1.
Platinum shadowing for correlative light and electron microscopy
Authors: Mateos Jose Maria , Guhl Bruno , Doehner Jana , Barmettler Gery , Kaech Andres , Ziegler Urs 
Published: December 2016

Fluorescence microscopy reveals molecular expression at nanometer resolution but lacks ultrastructural context information. Electron microscopy provides this contextual subcellular details but protein identification requires elaborate protocols. Correlative light and electron microscopy produces complimentary information that expands our knowledge of protein expression in cells and tissue. Even though a number of correlative approaches are currently available, few of these allow subcellular localization in tissue because of the challenges with sample preparation and 3D complexity. Tokuyasu cryo‐sections (Tokuyasu, 1980) preserve the sample ultrastructure and antigenicity of most epitopes, however, heavy metal exposure generates weak contrast to the samples rendering often interpretation of the data difficult.
We present a quick, simple and reproducible method for protein localization by conventional and super‐resolution light microscopy combined with platinum shadowing and scanning electron microscopy to obtain topographic contrast from the surface of ultrathin cryo‐sections collected on silicon wafers. Figure 1 shows protein distribution at nuclear pores in the topographical landscape of mouse kidney tissue.
New approach for low dose electron diffraction tomography
Authors: Tietz Hans , Sparlinek Peter , Olyenikov Peter , Ghadimi Reza 
Published: December 2016

Due to strong interaction of electrons with matter, 3D electron diffraction tomography has been proven to be a reliable method to solve structures of very small crystals, compared to X‐ray diffraction; even for small protein crystals [1].
Several procedures for automated acquisition of tomographic diffraction data sets have been described [2, 3], using precession technique or discrete beam tilt perpendicular to the goniometer tilt axis. However, those mentioned methods are mainly used for less beam sensitive materials and are not suitable for low dose applications.

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

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

X‐ray data have been measured with diffractometer D8 Advance (CuKa radiation, one‐dimensional detector Lynx–Eye with nickel filter). The range of the measurement was 2q = 10 – 120° with step 0,02° and acquisition interval 35,4 c. The program Topas 4.2 (Bruker AXS, Germany) and initial structural data of inorganic base ICSD, FIZ Karlsruhe, Germany have been also used in calculations.
X‐ray data analysis allows make conclusion that the surface modification of the soda lime silica glass by the surface ion exchange and chemical etching has a substantial influence upon the size of gold crystal grains (Figure 1 i). Gold crystal grains have elongated shape in direction . The increase of the gold nanolayer thickness leads to decreasing of the crystal grains size. The least size have been related to gold nanolayer 200 nm thick on glass surface modified by combined method indicated above: in direction average calculated grains size is ~ 14.7 nm; grains size averaged in directions , and is ~ 6.8 HM.
Authors express thanks to N.V. Bulina for technical support.
Power ofFIB‐SEMTomography for Biological Samples
Authors: Kizilyaprak Caroline , Niedergang Florence , Bellis Damien De , Blanchard Willy , Daraspe Jean , Geldner Niko , Humbel Bruno 
Published: December 2016

Three‐dimensional (3‐D) spatial distribution of organelles within cells at nanometer resolution is essential to better understand cellular processes and functions. Currently, FIB‐SEM tomography provides a promising technology to generate volume data with nanometer resolution (Bushby et al., 2011; Heymann et al., 2009; Knott et al., 2011; Villinger et al., 2012; Wei et al., 2012). A FIB‐SEM microscope is a scanning electron microscope (SEM) combined with a focused ion beam (FIB) where both beams coincide at their focal points. This combination enables to locally section bulk embedded‐resin samples by ion milling, creating a fresh surface for subsequent imaging with the electron beam. This process is repeated automatically to generate 3‐D information of relatively large volumes with a field of view of several micrometers.Any stained and embedded resin samples prepared for transmission microscope can be used for FIB‐SEM tomography offering the possibility to visualise directly in 3D a wide range of biological samples and typically volumes with a pixel size of 10 nm can be achieve (Figure1). However, to obtain a better resolution, considerations have to be given concerning ultrastructure preservation and samples preparation (Kizilyaprak et al., 2015). High pressure freezing (HPF) combined with freeze‐substitution (FS) and resin embedding constitutes a method of choice to find the best compromise between ultrastructural preservation and high contrast of cellular components (Figure2).In conclusion, we propose biological sample preparation protocols that can serve as starting point to visualize in 3‐D wide range of biological samples at nanometer resolution including HPF/FS samples and correlative microscopy approach using FIB‐SEM Tomography.
OptimizingCLEMprotocols for plants cells: A good preservation ofGFPfluorescence and ultrastructure in Arabidopsis roots
Authors: Marion Jessica , Bars Romain Le , Satiat‐Jeunemaitre Béatrice , Boulogne Claire 
Published: December 2016

While there is an explosion in Correlative Electron and Light Microscopy (CLEM) protocols, many have been developed on different eukaryotic models, though reports on plant cell exploration by CLEM remain scarce (Bell et al., 2013). Indeed, plant cells represent a challenge for imaging in many ways. Plant specificities have to be taken into account when optimizing CLEM protocols. To name some, strong autofluorescence of cellular components specifically present in plant cells (chlorophyll, pigments, etc.) and interfering with fluorescent signals of reporter proteins, presence of a large central vacuole complicating the fixation and the dehydration processes needed for TEM processing, cell wall and cuticles acting as physical barriers and impairing resin embedding processes (Hawes et al., 2001).Here we report two “in hand” robust and easily reproducible protocols to make such CLEM approaches on plant material. The proposed protocols had been worked out on the delicate question of autophagosomes identity in plant cells. Autophagy is a degradation process of intracellular components mainly implicated in response to environmental stresses. It consists in the delivery of cytosolic cargos entrapped by a compartment, named autophagosome, to lytic compartments for degradation and recycling. In plant cells as in any eukaryotic cells, autophagosomes have been identified as ring‐shape or punctate structures, thanks to the light imaging of reporter proteins of markers for autophagosomes (Le Bars et al., 2014).This study is using a Arabidopsis transgenic lines stably expressing GFP‐ATG8, which is a common marker for autophagosomes. Two CLEM protocols are used: firstly the high pressure freezing followed by acrylic resin embedding, and on the other hand the Tokuyasu method. Here we compare the performance of the two protocol to preserve GFP fluorescence and enhanced ultrastructural features in plant cells.
High Resolution Chemical Imaging on the Helium Ion Microscope
Authors: Dowsett David , Audinot Jean‐Nicolas , Vollnhals Florian , Eswara Santhana , Wirtz Tom 
Published: December 2016

The ORION NanoFab helium ion microscope (HIM) has become an ideal tool for imaging and nano‐patterning tool owing to its high lateral resolution [1]. Helium ions lead to resolutions of 0.5 nm for SE based imaging, while structures with sub 20 nm feature sizes may be rapidly patterned using Ne. Despite these advantages, the analysis capability of the machine is currently limited. At beam energies of 35 kV helium ions do not lead to the emission of characteristic X‐rays from a sample. While some compositional information can be obtained from back scattered helium [2], identifying elemental information is more difficult. Secondary Ion Mass Spectrometry (SIMS) is a powerful ion beam based technique for analyzing surfaces with high sensitivity and high mass resolution. SIMS is based on the generation and identification of characteristic secondary ions by irradiation with a primary ion beam (in this case helium or neon). The typical interaction volume for SIMS is around 10 nm in the lateral direction. As the probe size in the HIM is substantially smaller (both for He and Ne) the lateral resolution is limited only by fundamental considerations and not, as is currently the case on commercial SIMS instruments, the probe size [3‐4]. The prospect of adding SIMS to the HIM yields not just a powerful analytical capability, but opens the way for in‐situ correlative imaging combining high resolution SE images with elemental maps from SIMS [5].
We have developed a prototype SIMS spectrometer specifically adapted to the HIM. Notably the instrument is capable of producing elemental SIMS maps with lateral resolution limited only by the fundamental interaction between the primary beam and the sample. All elements/isotopes and small clusters with masses up to 500 amu are detectable with a mass resolution M/ΔM greater than 400 and parallel detection of 4 mass channels.
In this presentation we will introduce the HIM‐SIMS technique and present the latest application results in the field of materials science.
In vivo structural characterization of bacteriophage 812K1‐420 virion lytic cycle intermediates
Authors: Peterek Miroslav , Novacek Jiri , Plevka Pavel 
Published: December 2016

Staphylococcus aureus is one of the most important bacterial pathogens causing a wide spectrum of diseases varying in severity. Treatment of the infections is often complicated by S. aureus resistant to antibiotics. Current development of antibiotics is not fast enough to counterpart the emergence of antibiotic‐resistance. Preclinical studies showed, that phage therapy is promising alternative for treatment of bacterial infections including those caused by resistant strains, but approval of bacteriophage‐based drugs requires a detailed functional understanding of the molecular mechanisms of phage infection which we are still lacking.
Bacteriophage 812K1‐420 from the family Myoviridae is potential candidate to be used in phage therapy against S. aureus. Polyvalent and strictly virulent bacteriophage 812K1‐420 virion is composed of an icosahedral head containing linear 145,5 kb dsDNA, contractile tail and baseplate which specifically recognizes specific surface on the bacterial host. Replication and virion‐assembly mechanisms inside host cell are not fully explored yet, although few bacteriophage 812K1‐420 lytic cycle intermediates of virion assembly were reproduced in vitro.
Here we present initial structural analysis of bacteriophage 812K1‐420 infection in vivo using cryo‐electron tomography. We used cryo FIB milling technique to thin plunge frozen S. aureus cells (600‐1000 nm in diameter) infected by phage 812K1‐420 into wedge geometry . The cryoET data acquired on FEI Titan Krios microscope and tomograms were reconstructed using program IMOD.
How the Detector Geometry Influences
EDXS
Quantification
Authors: Kraxner Johanna , Schäfer Margit , Röschel Otto , Paller Manuel , Haberfehlner Georg , Kothleitner Gerald , Grogger Werner 
Published: December 2016

Thanks to the recent advent of very large area sensors and multi‐sensor systems X‐ray spectrometry in analytical electron microscopes received a substantial boost. In particular, the combination of 2 or 4 detectors has proven to obtain such a good sensitivity that the detection of single atoms is feasible [1]. However, if it comes to quantitative work partial or full shadowing of one or more detectors by the specimen holder will introduce systematic errors of the obtained results [2].
In this paper we closely investigate the influence of the detector‐microscope geometry on quantification using a ChemiSTEM system (Super‐X) on a Titan microscope with a low background double tilt high visibility holder [3]. We introduce a procedure, which allows for the first time to determine experimentally the detector positions inside the microscope chamber in terms of elevation angle, azimuthal angle, and distance. We measured the amount of shadowing as a function of specimen tilt angle α and compared these measurements to simulations, where we varied the detector positions in order to optimize the fit to the experimental data. The sample was a specially designed Au‐Pd thin film deposited on a silicon nitride membrane grid. The obtained positions were then verified by comparing the results to data obtained from different positions of the specimen holder.
To measure the amount of shadowing we used normalized intensities of the available X‐ray lines. An optimized simulation for one of the Super‐X detectors (Q1) may be seen in fig. 1 (Si‐K data). By comparing measurements from different lines we noted a significant difference depending on the X‐ray energies (compare Si‐K and Au‐L data in fig. 1). By further investigating this issue we found that the Beryllium specimen carrier inside the specimen holder may turn transparent for high enough X‐ray energies (e.g. Au‐L). In other words, the amount of shadowing not only depends on tilt angles and specimen positions but also on the energy of the X‐ray line of interest (see fig. 2).
Once the detector positions are known the shadowing effects can be taken into account and their influence on quantitative results can be corrected. In this paper we characterize potential effects and systematic errors they introduce, which are relevant to conventional EDXS work as well as analytical tomography experiments. The procedures described will transform EDXS on a multi‐detector system from qualitative analysis to full quantitative analysis.
Structural characterization of the human potassium channel Kir 2.1
Authors: Dezi Manuela , Wagner Renaud , Bendahhou Said , Venien‐Bryan Catherine 
Published: December 2016

Inwardly‐rectifying potassium (Kir) channels regulate membrane electrical excitability and K+ transport in many cell types where they control such diverse processes as heart rate, vascular tone, insulin secretion and salt/fluid balance. The physiological importance of eukaryotic Kir channels is highlighted by the fact that genetically‐inherited defects in Kir channels are responsible for a number of human diseases such as in Andersen's syndrome (Kir2.1), Bartter syndrome (Kir1.1), and neonatal diabetes (Kir6.2). To date, the available treatment is unfortunately not rational but rather empirical and this is mostly due to the lack of knowledge about atomic structure of these channels.
To elucidate how channel function becomes defective in the disease state requires a detailed understanding of channel structure in both the open and closed states. We have reported the structure of a homologous bacterian KirBac3.1 potassium channel with an open bundle crossing indicating a mechanism of channel gating determined by X‐ray crystallography at 3Å resolution. In this model, the rotational twist of the cytoplasmic domain is coupled to opening of the bundle‐crossing gate via a network of inter‐ and intra‐subunit interactions [1,2]. In addition, we have also used EM analysis of 2D crystals of the same Kirbac channel trapped in an open state and compared these results with the 3D structure [3].

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

Here we show preliminary results on the expression, the purification and the imaging of negatively stained isolated kir 2.1 in detergent. The particles have an averaged size of 12 nm consistent with a tetrameric form of the native channel. The oligomeric organisation is supported by native gel electrophoresis and Dynamic Light Scattering measurements.
Our results will hopefully contribute to uncover the mechanism of clinically‐relevant disease‐causing mutations on the structure, dynamics, and function of kir2.1 potassium channels and at investigating the potential of pharmaceutical correctors.
High‐resolution identification of immuno‐labelling nanoparticles on tissue using
X
‐ray detection
Authors: Scotuzzi Marijke , Kuipers Jeroen , Wensveen Dasha I. , de Boer Pascal , Hagen Cornelis W. , Giepmans Ben N G , Hoogenboom Jacob P. 
Published: December 2016

Cellular structure can be imaged at nanometer resolution using electron microscopy, but the biological molecules remain invisible in grayscale electron micrographs. With Correlative Light and Electron Microscopy (CLEM)[1], fluorescence recorded on a separate light microscope can be used to identify molecules in colour, but the resolution gap with electron microscope precludes accurate localization. The use of super‐resolution fluorescence microscopy techniques combined with CLEM[2,3] holds great promise but is still one order of magnitude off in resolution compared to EM. In addition, accurate registration of the separate images may be challenging and correlation may require expert procedures to maintain, e.g., protein photo‐switching under EM preparation conditions. Thus, while CLEM adds information represented in the colors of visible light, molecular identification and localization at the resolution of the electron microscope is still hampered. Therefore, we are exploring novel approaches to achieve color‐EM using the electron beam to add information about the specimen to the electron micrographs.
Detection of electron‐beam excited luminescence has been explored in the past. Cross‐sections for visible luminescence (also called cathodoluminescence) are, however, small and most biological fluorescent labels are destroyed before sufficient signal has been collected to allow high‐resolution localization [4]. Phosphorescent nanoparticles are considered as non‐bleaching alternative, but particle sizes are not yet well‐defined and bio‐functionalization has in most cases still to be pursued[5].
Here, we explore detection of X‐ray fluorescence to identify nanoparticle labels[6] at EM resolution in colour on tissue. We use Au and CdSe colloidal quantum dots, immuno‐targeted to guanine quadruplexes, resp. insulin, as molecular labels in large‐scale electron microscopy of pancreatic tissue. Energy dispersive detection of X‐ray emission (EDX) during electron irradiation allows discrimination of both nanoparticles based on their elemental (Au vs Cd) composition. We will present electron microscopy images overlaid with false colour elemental maps displaying single nanoparticles. Separate resolution tests have shown that individual Au particles down to 3 nm in size can be detected using EDX, highlighting the potential for coloured identification of materials in biological electron microscopy, revealing both compositional and ultrastructural information at nanometer‐scale resolution.