Many researchers have demonstrated reduced effects on

Many researchers have demonstrated reduced effects on quality or nutritional parameters including grapefruit juice [11], melon juice [12], apple juice [13,14], strawberry juice [15] and cranberry juice [16–18]. In order to observe changes of thermosonicated fruit juices and achieve inactivation of microorganisms, it is necessary to understand the mechanical, chemical, physical and sensorial changes in treated media. Thermosonication can be used to inactivate microorganism and specific attention is needed to avoid aromatic, sensorial and organoleptic changes in food. Today, am580 make demands on the quality, flavour and taste of different kinds of fruit juices [1,22,23]. It is important to keep very high quality standards of products including the taste and to make sure that juices and nectars meet predefined chemical and physical parameters. For this purpose, it is important to use sophisticated equipment and technical expertise in sample preparation and analysis and one of it are electronic nose and tongue [24–26]. In the study by Dias et al. [25] the electronic tongue was successfully applied for semi-quantitative discrimination of real juice soft drinks, based on the added fruit level. Non-specific lipo/polymeric membranes were am580 used for the first time, to perform quantitative determination of two major compounds present in those beverages, fructose and glucose. For sensory analysis, industry needs trained panellists, including a substantial amount of resources, time and cost [26–29]. It is important to quickly develop and test new methodologies that will ensure low-cost and reliable alternative to these costly and lengthy procedures. Electronic tongues of several types (potentiometric, voltammetric and impedance) may represent such alternatives. They have many applications and have been used to test several juices with a combination of a gas sensor array and voltammetric electronic tongue [13,25,27]. An electronic tongue functions by combining signals from non-specific and overlapping sensors with pattern recognition methods [25,26,30]. For an electronic tongue, the sample handling and delivery system is a trivial issue, and interfacing and conditioning circuits are handled by computer software. Our previous work was done on aromatic profiling of sonicated apple juice and usage of sonication in inactivation of microorganism [13,31].
The cranberry belongs to the same genus as the blueberry, Vaccinium. Cranberry has special combination of phenolic, proanthocyanidin, anthocyanin, flavonoid, and triterpenoid antioxidants [19–21]. There is special combination of three antioxidant nutrients in cranberry: resveratrol, piceatannol, and pterostilbene. The phytonutrients in cranberry provide maximal antioxidant benefits only when consumed in combination with each other, and also only when consumed alongside of conventional antioxidant nutrients present in cranberry like manganese and vitamin C.

Materials and methods

Results and discussion
For the treated samples of cranberry juice, according to the Table 1, cell plate is evident that for ultrasonic treatment B1.5 is invested the largest power (81W), and the lowest in the treatment of B1.9 (43W). The highest intensity of ultrasonic treatment was observed in samples B1.5 (63.94Wcm−2), and the lowest in the treatment of B1.9 (33.94Wcm−2). For the treated samples of cranberries nectar (Table 1), it is evident that for the treatment of B2.5 is invested highest power (82W) in the ultrasonic treatment, and the lowest in the treatment of B2.1 (43W). The highest intensity of ultrasonic treatment was observed in samples B2.5 (64.73Wcm−2), and the lowest in the treatment of B2.1 (33.94Wcm−2).

Conclusions
Principle component analysis (PCA) plot shows that some ultrasound-treated samples were different as sensed by electronic tongue compared to the untreated and pasteurised samples. Pasteurised samples are also different compared to untreated and ultrasound treated samples and this is qualitative difference. It can be concluded from the sensory evaluation that ultrasonically treated and pasteurised juices were evaluated with lower scores in comparison with the untreated samples. The best accepted are ultrasonically treated cranberry juice (sample B1.10) treated at the amplitude of 60μm for 6min and 40°C and cranberry nectar sample B2.6 treated at amplitude 120μm for 3min and 20°C. From the results in can be concluded that ultrasound and pasteurised samples were accepted from panellists. The scores were equal. This work demonstrates that sonication influences the aroma profile of cranberry juices and nectars, sensory properties and colour parameters. The process of thermosonication may be used to optimize critical process parameters to obtain juices that mostly retain original properties, and to be used as a possible alternative technique to pasteurisation.

The proposed piezoelectric wafer may be obtained

The proposed piezoelectric wafer may be obtained by means of cutting a type of single crystal, Pb(Mg1/3Nb2/3)O3–PbTiO3 (PMN–PT), in a special direction, shown as Fig. 1, in which [hkl] in crystallography denotes a direction and (hkl) a plane orthogonal to the direction [hkl]. The PMN–PT crystal first is orientated in a Cartesian coordinate system with crystal growth direction parallel to z axis. Then the PMN–PT crystal is poled in the [011] direction, and the wafer is cut in the plane (011). Zhang et al. [12] gives an example of this buy EDC.HCl piezoelectric constant matrix as following:where d36 is more than four times of d31 and d32. Generally, conventional d31 type piezoelectric wafer is able to excite Lamb waves (includes A0 and S0 modes) on the thin plate. Thanks to the shear deformation induced by d36, the proposed piezoelectric wafer is expected to excite both Lamb waves and shear horizontal waves.
When the piezoelectric wafer is used routinely, the direction of external electric field E is usually along the z-axis and is perpendicular to the surface of the wafer, which is bonded on the surface of plate structure. For the moment, only d36 is considered, and then Eq. (5) can be simplified as:
For a free d36 type piezoelectric wafer without in-plane external electric field and applied stress, the induced in-plane strain by applying a voltage across the z-direction can be expressed as:where is the thickness of the wafer. Fig. 1(b) shows the deformation of a d36 type piezoelectric wafer placed on the surface of a plate. The deformation can be considered due to a pair of shear strains, and . Moreover, the wafer can be equivalent to a group of line force, which is along the four edges of the wafer, applied on the plate. The induced pure shear deformation along the edges results in the guided waves in the plate.

Experimental investigations

Conclusions
This paper examines the fundamental characteristics of a new d36 type piezoelectric transducer, made from a PMN–PT crystal. Specifically the tuning frequency characteristics and directionality for the piezoelectric wafer are investigated in application of guided waves generation and sensing. According to the guided wave theory, the SH0 wave mode is a non-dispersive, which has many advantages for structural damage detection. The PMN–PT wafer has been demonstrated that it is capable of generating and detecting SH0 wave mode. Due to its materials anisotropy, the SH0 wave mode exhibits wave directionally. In the preliminarily FEM analyses, it proves above points based on the displacement components generated by the d36 type piezoelectric wafer, and then voltage responses show further that SH0 wave mode is dominant at 0°, but negligible small at 45°; while the A0 and S0 wave mode show the reverse trend.
Experiments were conducted to investigate both the tuning frequency characteristics and wave mode directionality for the proposed wafer. Based on wave amplitudes obtained, the following conclusions are drawn:

Acknowledgements
The financial support from the National Key Technology R&D Program under Grant No. 2011BAK02B02 & 2014BAG05B07, and the National Science Foundation of China under Grant No. 50908066 is gratefully appreciated.

Introduction
The thermal effects of delivering large amounts of acoustic energy through a focused ultrasound configuration in biological tissue (in vitro and in vivo) was first observed in 1942 [1]. Later on extensive animal studies [2] with focused ultrasound (FUS), demonstrated the reversibility of induced neurological dysfunction below certain temperature threshold and recognized the important technical and safety issues needed to overcome before employing the FUS in a clinical setting. The absence of imaging modalities capable of tissue temperature monitoring, delayed the development of FUS into a controlled, precise and safe tissue ablating therapy. The introduction of ultrasound and magnetic resonance imaging (MRI) with their fast technological advancement made image-guided focused ultrasound therapy a reality. The unsurpassed soft tissue contrast and its temperature sensitivity set MRI as the optimal imaging modality [3–5].

Elastic modules density film thickness and its

Elastic modules, density, film thickness and its changes for different values of humidity are determined from fitting the calculated and experimental data using corresponding equivalent circuits, Kirghoff’s equations and the least-square method [21,22]. The results of the theoretical analysis are also presented on Fig. 2. It has been found that unperturbed value of density ρ at RH=0.47% is equal to be 850kg/m3.
The change in the density Δρ/ρ due to humidity adsorption have not been measured by the method, but its maximum value ignoring mass of adsorbed water species is estimated from increase of the film thickness as Δρ/ρ=(hRH=0%–h)/h. This value is used in the paper to deduce the cholesterol absorption inhibitor contribution to the SAW humidity response.
Results of the measurements are presented in Table 1. They show that, the elastic modules of GO film are decreased and the film thickness is increased when humidity is grown up. The reducing the modules may be explained by penetration of the water molecules into interflake space and formation of H-binding between water molecules and groups C-OH, CO in GO film [7,11]. After reducing humidity to zero (last three lines of Table 1) the shear elastic module returns to its initial value within 30min, while the longitudinal one does not. This result may be explained by more fast desorption of water molecules from interflake space than from H-bindings.
Basing on data presented in Table 1, the SAW humidity response was evaluated using expression [23] connecting the SAW response Δv/vo with thickness h of a sorbent film, wavelength λ, electromechanical coupling coefficient k2, coefficients A, B, C dependent on the wave, and the changes in density Δρ/ρ, elastic modules ΔC11/C11, ΔC44/C44, conductivity Δσ/σ, capacitance per unit length CS2 and temperature ΔT produced in the film by a gas adsorption:
For particular case, when RH=67%, Δρ/ρ=−0.6, ΔC11/C11=−0.28, ΔC44/C44=−0.69 (Table 1), h=2μm, λ=105μm, the substrate is ST, x-quartz (Euler angles 0°, 132.75°, 0°), and A=2.85, B=1.26, C=0.26 [23], Eq. (1) gives that the part of response related with elastic contributions is equal to Δv/v0≈ 45ppm. At the same time, the SAW response measured experimentally for the same GO film, substrate material, and relative humidity by method [24] and network analyzer Keysight E5061B is turned to be one order of value larger: Δφ/φo=−Δv/vo=−550ppm (φ is a SAW phase detected by analyzer). Therefore, it was concluded that elastic variations in GO film are not dominant in the SAW humidity response. Other contributions could be originated from the changes either in film conductivity Δσ or temperature ΔT.
To clarify the role of conductivity the GO film was deposited both on weak (ST, x-quartz) and strong (128°Y-LiNbO3) piezoelectric substrates of the acoustic delay line (Fig. 3, Table 2). The sensitivities of the film towards humidity are evaluated from the changes in acoustic wave phases Δφ measured at room temperatures (20°C) and central frequencies f (Table 1) using a network analyser (HP 8753E, Agilent Technologies, Santa Clara, CA) operating in phase format. In order to avoid dependence of the measurements on the distance between transducers (LIDT) and frequency (f) the measured Δφ are normalized to the total phases φo=2πL/λ=2πLf/v acquiring the waves from input to output transducers [4]. In order to avoid dependence of the measurements on the length of GO film (LGO) the values Δφ/φo are normalized to (LIDT/LGO). As a result, the normalized responses S=(Δφ/φ0)×(LIDT/LGO) could be compared with each other for different waves and various substrates as determined at identical experimental conditions.
The quartz-based structure is measured to have much lower sensitivity than the lithium niobate-base counterpart. For example, for RH=18% the normalized SAW responses were S=−376 and −1440ppm, respectively. Therefore, we conclude that it is just the change in GO film conductivity that is responsible for the SAW humidity response most of all. This conclusion agrees with the other paper [16] where S0 Lamb wave having large coupling constant k2 demonstrated higher humidity response than A0 wave having small k2[25,26].

From our experimental observation we deduce

From our experimental observation we deduce that water molecules, each with a molecular mass of 18Da, move an RMS distance of ∼1Å for each e−/Å2. A typical cryoEM exposure having 25e−/Å2 will therefore result in a RMS displacement of water molecules by ∼5Å. In the same way as the thermal motion of water molecules results in Brownian motion, the beam-induced motion of water molecules will also be expected to displace embedded protein molecules. In a 25e−/Å2 exposure a protein molecule of 100kDa, such as hexokinase, embedded in this film of randomly diffusing molecules would be expected to have an RMS displacement of ∼1.0Å (hexokinase with a of 100kDa has a 20–40× smaller purchase Cy5.5 hydrazide coefficient than water, and so should move ∼4–6× less, according to the Stokes-Einstein equation for diffusion [19] in which the diffusion coefficient varies as ). For a protein molecule of 25kDa, the beam-induced random motion would be higher at ∼1.5Å, and for a ribosome of 2.5MDa somewhat lower at ∼0.7Å. This additional random motion produces image blurring that can be represented by an extra B-factor, which for hexokinase would correspond to ∼25Å2, on top of that resulting from other effects such as intrinsic radiation damage to the macromolecular assembly. We can thus conclude that random, Brownian type of beam-induced motion of biological structures is unlikely to be one of the limiting factors in attaining high-resolution structures using single particle cryoEM approaches. Only for very small particles at resolutions of 2Å or beyond is this type of beam-induced motion likely to be a fundamental limitation.

Acknowledgments
The authors acknowledge funding from the Medical Research Council, Grant no. U105184322. We would also like to thank Garib Murshudov and Nigel Unwin for their helpful comments.

Introduction
Since the sixties, quartz tuning forks (QTF) have been widely used in watches and other electronic devices as frequency standard mainly because of the stability of their mechanical properties with temperature fluctuations [1,2]. More recently, their high resonance frequency, quality factor and spring constant as well as their self-sensing capacity, thanks to the piezoelectricity of quartz, motivated their implementation in many scanning probe microscopy techniques such as scanning near field microscopy [3,4], scanning near-field acoustic microscopy [5] and magnetic force microscopy [6,7]. This has made QTF based sensors a very important tool for different scientific communities [6].
QTF were also implemented in atomic force microscopy (AFM) to serve as both actuator and sensor for tip-sample interactions which eliminated the need for optics and allowed low oscillation amplitude operations [6]. However, the gluing of a metallic tip to the QTF and the interactions with the surface induced break of the QTF symmetry resulting in low scanning speed and resolution. The qPlus sensor (QPS) introduced by Giessibl [8] gave a simple and efficient solution for these problems. In this design, one of the QTF prongs is firmly fixed to a supporting structure (ceramic) and a metallic tip (usually tungsten) is glued to the free prong using epoxy [1,8]. In this configuration, only one prong is deflected during scanning of a surface and the QTF is behaving essentially as a self-sensing cantilever which makes fast scanning possible and easy to interpret [1,8]. Since its introduction, the QPS attracted increasing attention and atomic resolution at low temperature in non-contact AFM (NC-AFM) mode is now routinely achieved by different groups [8–10].
Despite their wide use and commercialization, QPS are still handmade and suffer from decreased resonance frequency and quality factor when compared to the bare QPS (without tip) due to the mass load induced by the relatively large and heavy tungsten tip fixed to the free prong using conductive epoxy [8,2,11,12]. Moreover, it is impossible to know exactly the added mass, the amount of epoxy used and the position of the tip on the free prong. These imprecisions result in a large spread of the mechanical properties from one fabricated sensor to another and make it very difficult to calibrate the spring constant (stiffness) [12]. Even the type of epoxy used was shown to induce significant difference [13].

As mentioned previously this analysis applies to slices greater than

As mentioned previously, this analysis applies to slices greater than 10 times the knife radius. This is because the curved knife edge changes the effective knife angle during the cutting process. In order to estimate the forces for ultra-thin slices (<30nm), the rake angle was estimated from the following relationship:where r is the radius of the diamond knife edge. From this relationship, with 3nm thickness slicing, the rake angle becomes 0°. The TIC10 of the thickness increases the effective rake angle and, consequently, reduces the shear angle. Thus, selecting very thin slices increases the thrust and friction forces, which lead to an increase in damage to the block face. Slice thicknesses of 2.5nm have been reported [27], whereas sectioning below 2.5nm has not yet been successful. This might indicate the need for a minimum shear angle or maximum knife angle to enable the material to be sliced. In such cases, the thrust and friction forces may become greater than the cutting force and, consequently, the knife edge would rub along the block face instead of cutting it. Further, the slicing involves plastic deformation ahead of the diamond knife tip and elastic recovery of the block face after the knife has passed, resulting in a slight upward movement of material at the block face near the diamond knife tip. Importantly, the shear plane appears to start at a point above the diamond knife tip due to an increase of the thrust force as a consequence of reduction of the rake angle. Fig. 8 shows a schematic diagram of the force relations whilst taking account of the influence of the radius of the diamond knife. We assume that the difference between the thicknesses of the shear force starting point and the recovering layer is an atomic layer, as indicated by the following relationships:where a is the thickness of the recovering layer, ω is the angle between the shear force starting point and the cutting direction on the edge of knife, and β2 is the angle from the perpendicular to the cutting direction on the knife tip and the recovering layer touching the knife tip. The shear angle can then be determined from the following relationship:

Optimising the cutting conditions
The most important strategy for optimising the cutting conditions is to reduce the forces applied to the block face since these lead to the deformation. The stress at the block face arises from a combination of the cutting force, Fc, and the normal force, Ft, as indicated in the following expression:
This stress is strongly influenced by the rake angle. Fig. 9 shows the relationship between the compression rate, the shear and rake angles, and the applied stress. Reduction of the rake angle results in an increase in the shear angle and a reduction of the stress. When, the rake angle is 50°, the shear and normal stresses become equal, while for a rake angle of 76° the normal stress becomes zero. Consequently, if the knife and clearance angles sum to 14°, the damage on the block face is minimised. Further, when the normal stress exceeds the ultimate tensile strength the chip will form by ripping rather than by slicing. When the rake angle exceeds 50°, the normal stress becomes less than the tensile yield stress of the AA 2024 alloy. Hence, damage to the block face might be reduced. However, it is also dependent on the dislocation density, which is readily influenced by deformation on the block face.
Currently, the minimum rake angle of a commercially available diamond knife is 35° however, it is possible to minimise the normal stress. In order to minimise the stresses, the knife can be oscillated to minimise the apparent knife angle [46]. The apparent knife angle (mean knife angle during the oscillation of the knife), α2, is given by the following equation:where vs is the traverse speed of the knife and ve is the velocity normal to the cutting direction. The apparent angle can be applied in the Merchant cutting model and it determines the ultramicrotomy operating conditions for minimum damage on the block face. Notably, the oscillating knife reduces the apparent angle and, thereby, the associated stresses. Additionally, the transverse movement of the knife also causes the local temperature of the material in contact with the knife edge to increase because of the friction between the knife edge and the material, causing the material to soften. The effect of oscillation on reducing chatter and generally improving the surface finish is evident in Fig. 10.

Of course it is apparent that the indications identified

Of course it is apparent, that the indications identified in the present test experiment regarding secondary and inelastically scattered Chloroquine momentum mapping as a possible source of information, are not sufficient for a detailed and theoretically complex analysis of scattering processes. However we hope to have demonstrated an interesting capability of our cathode lens based spectromicroscope equipped with “in-lens” electron sample illumination system for near simultaneous, energy selective real imaging and “energy-loss”/secondary electron momentum mapping, and therefore for investigating the different scattering phenomena. They are intended to be the subject of further, more detailed experimental and theoretical studies exploiting the electron energy-loss (plasmon) imaging and momentum mapping in the high energy resolution regime. It\’s importance for the band structure analysis and -imaging in the case of electron photoemission [43,44] has been demonstrated recently by the application of full-field high resolution momentum microscopy: (1) time-of-flight imaging of the d-like surface resonances on Mo(110) [45] and (2) spin resolving high resolution energy selective imaging of the momentum distribution of photoelectrons [46].

Acknowledgments
I would like to express my gratitude to Professor Ernst Bauer for his encouragement and stimulation to this experiment, to Janusz Krajniak for his invaluable R&D work on the electronics and software, to Jerzy Dora for his unique concepts in electronics, to Bartosz Czaban for the same in imaging software and to Dariusz Mirecki for discussions and help. Financial support from the National Centre for Research and Development in Warsaw under Grant No. INNOTECH-K1/H11/26/159076/NCBR/12 is gratefully acknowledged.

A crucial step towards the optimization of the

A crucial step towards the optimization of the optoelectronic device structures is the investigation of the defect formation mechanisms. Hence, the detailed atomic study of the local structure at the interface between the semiconductor epilayers and the Si substrates is of particular interest. For this, transmission electron microscopy (TEM) is ideally suited as it provides the spatial resolution to study the structure of materials on the atomic level. Over the past 30 years, extended defects in semiconductor materials have been intensively studied by conventional and high-voltage high-resolution TEM [1,9–12]. However, it was experimentally challenging to resolve the exact atomic-scale structure due to the lack in spatial resolution and/or image delocalization in conventional phase contrast micrographs. Thus, “holographic” reconstruction techniques were indispensable to increase the spatial resolution [13–15]. Alternatively, image simulations based on structural models were performed to validate the image interpretation [11,12].
More recently, the implementation of spherical-aberration correctors in scanning transmission electron microscopes (STEM) [16–18] has lead to dramatic improvements in lateral resolution. These instruments are now capable of routinely producing images in the deep sub-Ångström range [19]. Thus, the dumbbell structure in crystalline Si along and samples can be clearly resolved, which is particularly useful for the characterization of defects [20]. Additionally, by using a high-angle annular dark-field (HAADF) detector it is possible to produce images which show contrast that approximately scales with the square of the atomic number, hence the name Z-contrast imaging. This allows to discriminate, for example, between the lighter Cd atomic columns from the heavier Te columns in the CdTe dpn semiconductor. Recent studies of low-dimensional semiconductor structures and devices by aberration-corrected STEM clearly demonstrate the power of this technique for investigating defects and interfaces [3,21–29]. It has proven to be decisive both in the detection of novel types of defects but also in the advancement of our understanding of seemingly basic crystal-structure defects.

Experimental details
Unless otherwise stated, all experimental images were taken using a double spherical aberration-corrected JEOL JEM-ARM200F microscope equipped with a cold field-emission electron source operating at 200kV. In STEM mode, a convergence semiangle of 25mrad was used in combination with an annular dark field (ADF) detector with inner and outer collection semiangles of 90 and 370mrad, respectively. A Gaussian low-pass filter for noise reduction was applied to all images.
Samples for the HAADF-STEM analysis were prepared by means of either a FEI Helios NanoLab 600i or FEI Helios NanoLab 450S focussed ion beam (FIB) operated at accelerating voltages of 30 and 5kV. Additionally, Si/Ge specimens [30] were prepared by mechanical polishing and dimple grinding, followed by ion-milling with Ar+ ions using a Fischione Model 1050 TEM-Mill operating at low voltages and grazing incidence to achieve electron transparency.

Configuration of planar defects
The most commonly observed planar (2D) defects in cubic semiconductors by using high-resolution TEM are twin boundaries (TBs) and stacking faults (SFs) [31]. They are caused by discontinuities in the …AaBbCcAaBbCc… stacking sequence of 111 -type close-packed layers in diamond or, its ordered variant, zincblende structure. Additionally, compound semiconductor layers grown on elemental semiconductor substrates, like Si or Ge, are also susceptible to form antiphase boundary (APB) defects. In the following sections, the characteristics of these defects are discussed.

Configuration of dislocations
Fig. 4 illustrates the presence of dislocations both inside a Ge crystal and at a Ge/Si interface, together with their corresponding ε strain field maps obtained by geometrical phase analysis (GPA) [53]. In particular, Fig. 4a and b show two parallel dislocations lying on perpendicular slip planes in close proximity within a Ge crystal and the strain field interaction between them: both dislocation cores exhibit a compression region (in blue) and a tensile region (in yellow) [30]. Similar butterfly-like shapes are observed at the Si/Ge interface (Fig. 4c and d). They are MDs (marked with white arrows) resulting from the 4.2% lattice mismatch between Ge and Si, and are identified as pairs of perfect glissile 60° and perfect sessile 90° MDs (a more detailed description of these type of dislocations is given in the following sections). The number of atomic planes between the MDs is not constant. It varies between 20 and 40 planes depending on the area of observation. Theoretically, for the total relaxation of the misfit strain one MD should be introduced every 24 planes [, n=number of planes, a=lattice constant, a=0.5658nm and a=0.5431nm]. Experimentally, the number of 111 planes between the MDs is slightly larger with an average of 26±0.5 planes between dislocations. Therefore, not enough MDs are incorporated to fully relax the strain plastically at the selected growth temperatures [54].

To test the feasibility of determining defocus and astigmatism within

To test the feasibility of determining defocus and astigmatism within the error limits discussed above from the diffractogram of the side band image I used the tool CTFIT from the EMAN1 package [9]. Based only on the first zero I determined a defocus of about 195nm and an additional defocus in the y-direction of about 45nm (rather than 200 and 40nm). I did not try to evaluate the accuracy of the direction of astigmatism. Obviously, due to the nature of the diffractogram, it\’s not possible to use an existing automatic procedure for determining the CTF-parameters.

Conclusions
I present a novel design for a single side band aperture which allows determining defocus and astigmatism from the recorded images. I present the image formation theory and a method of correcting the transfer function of such an imaging system and test both in simulations. One clear advantage of single side band imaging is that the modulus of the complex valued transfer function is 1 for all spatial frequencies (outside the gaps). This means that images can be recorded at an arbitrary defocus value without introducing zeros in the transfer function. Therefore single side band imaging with such an aperture could be very suitable for imaging small proteins (100kDa and below). Thus this imaging mode could be an attractive alternative to imaging with phase plates.

Acknowledgement

Introduction
Strongly correlated MLN4924 materials exhibit various intriguing and drastic phenomena such as the metal-insulator transition, high- superconductivity, and colossal/giant magneto resistance [1]. When multiple phases are adjacent and competing in the vicinity of a first-order phase transition for instance, there emerges self-organized electronic inhomogeneity with various types and length scales [2]. In order to find deeper insight into the macroscopic phenomena in these materials, it is indispensable to understand local electronic structures with relevant spatial resolution.
Scanning photoemission microscopy (SPEM) is one of the primary spectroscopic techniques for studying electronic states with spatial inhomogeneity [3]. Recently, this type of instrument has been developed to incorporate the capabilities of angle-resolved photoemission spectroscopy (ARPES), mainly at third-generation synchrotron facilities, often referred to as µ-ARPES or nano-ARPES [4–6]. These techniques have successfully revealed various electronic inhomogeneities in strongly correlated electron materials, such as metallic and insulating phase separation with a length scale of 10 µm in Cr-doped V2O3[7], electronic and structural inhomogeneities with a length scale of 100 µm in high- cuprate YBa2Cu4O8[8] as well as bilayer manganites LaSrMn2O7[9]. However, it is often inevitable to lower energy resolution to get higher count rates since photon flux is considerably reduced to achieve high spatial resolution due to low efficiency of focusing optics.
On the other hand, a major focus of conventional ARPES has been to study the density-of-states, band-dispersions, and Fermi surfaces of solids [10]. Modern ARPES with high energy and momentum resolutions, usually referred as high-resolution ARPES, can precisely determine quasiparticle\’s dispersion relations and lifetimes [11,12]. However, detailed microscopic information in the smaller area has not been sufficiently pursued by means of high-resolution ARPES so far.
By maximizing the merits of high energy and spatial resolutions, we have developed a new laser-based µ-ARPES system at the Hiroshima Synchrotron Radiation Center (HiSOR). High brilliance and monochromaticity of laser light is suited for high-resolution ARPES [13–15], and furthermore, its spatial coherence/directionality can be applicable to SPEM. In this paper, we present the design and typical performance of our µ-ARPES system equipped with a vacuum ultraviolet (VUV) laser tunable from 5.90 eV to 6.49 eV. Based on considerations on the commercially achievable specifications of laser light source, optics and focusing systems, we realized the compatibility with high spatial resolution better than 5 µm as well as the state-of-the-art energy and momentum resolutions. We have also examined spatial dependence of fine spectral features, which enables us to find sample area suitable for obtaining intrinsic electronic states. Present µ-ARPES holds the promise for uncovering intrinsic and fine details of electronic features that may have been overlooked by conventional high-resolution ARPES.

The production and accumulation of the virally encoded

The production and accumulation of the virally encoded proteins signals a switch in the polymerase function, from viral mRNA transcription to genome replication, in which N plays a critical role. An essential step in the viral replication of the nascent positive-sense genome (antigenome) relies on its encapsidation, a process facilitated by cis-acting conserved sequences located on the 3′ ends of viral genome and antigenome (Whelan and Wertz, 1999; Li and Pattnaik, 1999). Additionally, N and P proteins are critical in promoting genome replication, as the N/P complex provides the structural and chaperone support for the nascent RNA to bind via sugar-phosphate interactions to the N protein (Albertini et al., 2006). The bound antigenome will then function as template for the synthesis of encapsidated negative-sense genomes, which will be assembled into progeny virions.
Virion assembly is a staggered process where the various components [nucleocapsid core (RNP), G and M proteins] are sequestered in different cellular compartments and converge in the final steps of the process. The nucleocapsid is assembled during RNA replication in the cytoplasm, as is observed for members of the genera Vesiculovirus, Lyssavirus, Ephemerovirus and Novirhabdovirus. Viral G protein is inserted into the endoplasmic reticulum where chaperones (BiP and calnexin)(Hammond and Helenius, 1994) facilitate its proper folding and assembly into trimers (Doms et al., 1988), prior to transport and fusion into the Golgi complex. As it traffics through the cell it undergoes further posttranslational modifications including glycosylations (Schmidt and Schlesinger, 1979), prior to its transport to cholesterol- and sphingolipid-rich lipid rafts in the baso-lateral plasma membrane. M protein is synthesized mostly as a soluble protein in the KN-93 hydrochloride (McCreedy et al., 1990) and is also membrane bound, albeit at lower amounts (Ogden et al., 1986). However both forms of the M protein are recruited for assembly of nucleocapsid/M complexes at the host plasma membrane from where virions will bud (Odenwald et al., 1986). This budding process is facilitated by the interaction of M with host-encoded proteins responsible for the formation of multivesicular bodies (MVB), and their release from the plasma membrane (Harty et al., 2001).

‘Classical’ vertebrate rhabdoviruses
For historical reasons any reference to classical vertebrate rhabdoviruses denotes members of the genera Vesiculovirus and Lyssavirus, represented by the prototype species vesicular stomatitis Indiana virus (VSIV) and rabies virus (RABV), respectively. Vesiculoviruses have a wide host range among mammals and are transmitted by hematophagous insects (sandflies and/or mosquitoes). Lyssaviruses utilize mostly bats as their principal reservoir hosts as well as various terrestrial carnivores as terminal hosts. Viruses of each genus form a monophyletic clade in a maximum likelihood (ML) tree inferred from complete L protein sequences (Dietzgen et al., 2011; Walker et al., 2015). Structurally both demonstrate the classic rhabdovirus enveloped bullet-shaped virions (Fig. 1) packaging a genome consisting of five genes (3′-N-P-M-G-L-5′), each separated by a short gene junction (intergenic region), and flanked by highly conserved 3′ leader (le) and 5′ trailer (tr) sequences (Fig. 3). In vesiculoviruses the P gene mRNA contains 2 additional alternate start codons that initiate translation at alternative open reading frames (ORFs) that encode two small basic proteins C and C’ (55-aa and 65-aa, respectively) of unknown function (Spiropoulou and Nichol, 1993; Peluso et al., 1996). Suppression of C/C’ expression has no apparent effects in virus replication or pathogenicity in vivo (Kretzschmar et al., 1996). Of note is that not all members of the genus express alternative ORFs in P [e.g. vesicular stomatitis Alagoas, Maraba, Malpais Spring, Morreton viruses] (Walker et al., 2015), and additional ORFs KN-93 hydrochloride (≥150nt) may be present in alternative reading frames in other genes than P (Walker et al., 2015).

The aim of the present

The aim of the present study was to assess the effect of different inflammatory stimuli on eBM-MSCs immunoregulatory ability and immunogenicity, studying the expression of immunogenic and immunomodulation-related molecules. Firstly, the influence of allogeneic inflammatory SF on eBM-MSCs was investigated, and subsequently, the effect of priming eBM-MSCs with a combination of the two pro-inflammatory molecules IFNγ and TNFα, was tested at two different doses. SF and CK inflammatory conditions were chosen according to previous studies (Ren et al., 2008; Leijs et al., 2012; van Buul et al., 2012; Vézina Audette et al., 2013; Zimmermann and McDevitt, 2014). This work contributes to understand the effects of inflammatory exposure on eBM-MSCs, as a previous step to enhance their use in vivo for equine joint diseases.

Materials and methods

Results

Discussion
The cultured SEA0400 showed capacity for attachment to plastic and the ability of differentiation into osteoblast, adipocyte and chondrocyte, as criteria established to define human MSCs (Dominici et al., 2006). Equine BM-MSCs displayed a gene and cell surface expression pattern similar to previous reports for this species and showed a normal growth pattern, with a proliferation rate and viability similar to other studies (Ranera et al., 2011). Inflammatory conditioned media were prepared based on recent publications on this field. Inflammatory SF was obtained from an allogeneic donor and added at a concentration considered likely to act as an enhancer of the immunoregulatory potential of MSCs (Leijs et al., 2012). SAA and Hp were used to determine the inflammatory status of the SF. Both APP were elevated with regard to described ranges (Basile et al., 2013; Jacobsen et al., 2006) in agreement with the expected changes for these proteins (Jacobsen and Andersen, 2007). For CK-conditioned media, the synergy displayed by the pro-inflammatory cytokines TNFα and IFNγ (Zimmermann and McDevitt, 2014) supported the decision of using them to stimulate the eBM-MSCs. The induction of immunoregulatory properties of BM-MSCs by synergistic cytokine priming is not previously reported in horses. Equine BM-MSCs has been stimulated with 100ng/ml of IFNγ alone in some studies (Paterson et al., 2014; Schnabel et al., 2014), but their gene and surface expression of immunoregulation-related molecules were not studied after the stimulation. Therefore, the tested doses (20ng/ml and 50ng/ml) were chosen according to previous studies in human and mouse MSCs showing induction of immunoregulatory factors expression or secretion (Ren et al., 2008; Waterman et al., 2010; Hegyi et al., 2012; van Buul et al., 2012) to determine if the same conditions also operate similar changes in the behaviour of MSC from equine species. The available volume of inflammatory SF did not allow a time course to be performed and thus a single time-point of 72h was chosen to maximize potential effects of exposure to SF. This was longer than previous studies on the effects of inflammatory SF (Leijs et al., 2012; Vézina Audette et al., 2013) but longer exposure to individual cytokines has been reported (Paterson et al., 2014) and immunosuppressive effects of IFNγ-activated MSCs have been shown to depend on the exposure time to IFNγ (Chan et al., 2006). Our results are similar to previous reports in other species using shorter times of cytokine stimulation, as it will be further discussed. However, the exposure of eBM-MSCs to SF did not produce remarkable effects despite of using an exposition time longer than the previously described (Leijs et al., 2012; Vézina Audette et al., 2013).
Several mechanisms have been proposed to participate in the immunoregulatory function of MSCs, including the participation of chemokine axis, adhesion molecules and soluble factors (Ma et al., 2014). The co-culture of MSCs in the presence of T cells triggers the expression of VCAM-1 (Ren et al., 2010), according to the results obtained in our CK20 conditions. This finding supports the implication of cell-to-cell contact in the MSC immunoregulatory mechanism. Adhesion molecules are also related to MSC migration, a mechanism which could be critical for the recruitment of MSCs into wound sites for tissue regeneration. This process is complex, especially in inflammatory environments, and whereas some studies describe the enhancement of MSC migratory property under inflammatory stimulation (Ries et al., 2007; Shi et al., 2007), others report a decrease in this property depending on the time of exposure (Waterman et al., 2010). The MFI of the hyaluronan receptor CD44, related to cell migration through the extracellular matrix (Zhu et al., 2006), was increased under CK50 conditions, indicating an enhancement in the expression level of this marker, despite the number of positive cells remained similar. However, the gene expression of the chemokine receptor CXCR4, also implied in the MSC migration (Honczarenko et al., 2006), was decreased in both CK Experiments, suggesting that MSC migration might be diminished in the tested inflammatory conditions.