Characterization of porcine oviductal epithelial cells, cumulus cells and granulosa cells-conditioned media and their ability to induce acrosome reaction on frozen-thawed bovine spermatozoa

The mammalian female reproductive tract cells have been wildely used in in vitro fertilization. We studied the secretory proteins of porcine oviductal epithelial cells (POEC), and cumulus cells (CC) co-cultured with granulosa cells (GC) in conditioned media (CM), and their ability in inducing acrosome reaction (AR) on frozen-thawed bovine spermatozoa. POEC and CC+GC were cultured in M 199 medium for 48, 96 and 144h prior to investigation of protein secretion. The results from SDS-PAGE showed secretory proteins sizes of about 17, 22 and >220kDa in both CM from POEC and CC+GC. To test the ability of CM from POEC and CC+GC in inducing AR, the CM was frozen for 1-3 months before incubating with frozen-thawed bovine spermatozoa. Percentages of the spermatozoa with AR were determined under an inverted microscope and it was found that the tested group 1 (incubated with fresh and frozen CM from POEC for 1-3 months) were 78.44+/-7.25, 75.78+/-4.41, 65.22+/-5.59 and 50.56+/-6.25, respectively. The tested group 2 (incubated with fresh and frozen CM from CC+GC for 1-3 months) had the percentages of the spermatozoa with AR at 88.67+/-4.03, 82.22+/-3.46, 71.00+/-3.16, and 58.56+/-4.69, respectively. Statistical analysis of all group percentages indicated that they were significantly different (P<0.05). Transmission electron microscope studies demonstrated that there were morphological changes on the sperm heads which resulted in the leakage of acrosin and subsequent AR. We concluded that the CM from both POEC and CC+GC efficiently increased in vitro AR on frozen-thawed bovine spermatozoa. Identification and functions of these secretory proteins are currently under investigation which may lead us to a better understanding of other beneficial effects in the utilization of both CM conditions.     

The liquid-phase adsorption of n-octylamine onto the graphite basal surface studied by tapping-mode atomic force microscopy

Solid-like structures formed on the graphite basal surface following the liquid-phase adsorption of n-octylamine have been studied using tapping-mode atomic force microscopy. Following deposition of a 1 μl droplet and subsequent annealing at 100°C, the amine formed randomly distributed islands categorised into two types based upon the morphology at the vapour interface. Evidence was found for the parallel orientation of the molecular axis at the basal plane, the orientation anticipated from studies of other aliphatic molecules. The results suggest the formation of vertically oriented molecular clusters at the vapour interface. Similarities were found with previous results of the adsorption of n-alkanes at the basal surface, highlighting the importance of n-alkyl chain interactions. Similarities and differences were observed between amine and alkane behaviour at the graphite steps. Annealing at 200°C reduced the island coverage, particularly at steps, and at 300°C no decoration was observed on the surface. The activation energy for surface diffusion and the energy difference between surface and vapour molecules are estimated. Upon deposition of a 5 μl droplet of amine onto graphite, an aggregate morphology decorated terraces and steps. Measurements suggest that the aggregate surface consisted of molecular clusters oriented towards the surface normal.     

Time-resolved photoluminescence study of the red emission in nanoporous SiGe alloys

Porous Si_1−xGe_x (PSiGe) layers with efficient room temperature visible photoluminescence (PL) were elaborated by anodical etching from p-type doped epitaxial layers with Ge contents from 5 to 30%. The luminescence is characterised by a broad PL band centred at 1.8 eV. Time resolved photoluminescence decay is studied in porous silicon germanium as a function of germanium content, temperature, emission energies and surface passivation. The PL decay line shape is well described by a stretched exponential in all cases. The effective lifetime at low temperature in as prepared porous Si_1−xGe_x is 400 μs, i.e. an order of magnitude less than in porous silicon. After the formation of a 20 Å thick oxide surface layer we observe a decrease of the effective lifetime to 20 μs at T=4 K.     

Effects of porcine follicle stimulating hormone, luteinizing hormone and estradiol supplementation in culture medium on ultrastructures of porcine cumulus oocyte complexes (pCOCs)

Inhibition of primary oocyte developing to secondary oocyte results from oocyte maturation inhibitor (OMI) which is secreted from oocyte-surrounding cumulus cells (CCs) during forming complexes with an oocyte. Development of primary oocyte occurs when the CCs are dissociated from the oocyte. This research studied the effects of pFSH, LH, and estradiol supplementation in culture medium on ultrastructures of porcine cumulus oocyte complexes (pCOCs) using transmission electron microcopy (TEM) and inverted microscopy. A total of 880 oocytes were isolated from 110 ovaries: an average of 8 oocytes per ovary. The oocytes were round in shape and surrounded by zona pellucida with layers of cumulus cells (CCs), at a diameter ranging between 90 and 150 μm and more than 400 μm. Based on the CCs surrounding an oocyte, pCOCs were classified into 5 types, which were intact-, multi-, partial cumulus cell layers, completely denuded oocyte, and expanded cumulus cell layer, which were found at the percentage of 53.86%, 14.32%, 4.32%, 19.20%, and 8.30%, respectively. The Types I and II pCOCs (intact- and multi-CCs layers) were further cultured at 37°C with 5% CO(2), 95% air atmosphere, and high humidity for 24-48 h to investigate their morphological changes after hormonal induction. For the pCOCs cultured without hormonal induction, the CCs were still round in shape and remained in contact with an oocyte via a process of granular end point sticking into the zona pellucida. In contrast, for the hormone supplemented groups, morphological alteration of pCOCs were seen after culture of 24-48 h. The CCs shape was changed from round into elongated or columnar in an opposite direction from an oocyte as well as no communication between microvilli of CCs observed. This led into ceasing of OMI secretion. Therefore, changes of CCs morphology were a sign of the beginning of oocyte maturation. Further study is to characterize the granular substance at the end point of CCs that stick into the zona pellucida.     

Tight-binding study of ammonia and hydrogen adsorption on magnetic cobalt systems

The semi-empirical self-consistent tight-binding model of ammonia and hydrogen adsorption at Co(0001) and small Co clusters is used to study the chemisorption role in surface magnetism. The adsorbate choice has been suggested by recent experiments. At the Co(0001) surface the atomic magnetization is predicted to diminish locally by 0.26 μ_B due to an isolated hydrogen atom adsorption; for Co₁₃ clusters the change is somewhat smaller but less localized. At H(1×1)–Co(0001) the magnetization of surface Co atoms drops to 0.88 μ_B. The hydrogen magnetic moment is very small and couples antiferromagnetically to Co. Ammonia adsorption is found to reduce the Co atom magnetization locally by 0.1 μ_B or less. We discuss the possibility of adsorbate–metal antiferromagnetic coupling in more detail.     

Scanning probe microscopy study of exfoliated oxidized graphene sheets

Exfoliated oxidized graphene (OG) sheets, suspended in an aqueous solution, were deposited on freshly cleaved HOPG and studied by ambient AFM and UHV STM. The AFM images revealed oxidized graphene sheets with a lateral dimension of 5–10 μm. The oxidized graphene sheets exhibited different thicknesses and were found to conformally coat the HOPG substrate. Wrinkles and folds induced by the deposition process were clearly observed. Phase imaging and lateral force microscopy showed distinct contrast between the oxidized graphene and the underlying HOPG substrate. The UHV STM studies of oxidized graphene revealed atomic scale periodicity showing a (0.273 ± 0.008) nm × (0.406 ± 0.013) nm unit cell over distances spanning few nanometers. This periodicity is identified with oxygen atoms bound to the oxidized graphene sheet. I(V) data were taken from oxidized graphene sheets and compared to similar data obtained from bulk HOPG. The dI/dV data from oxidized graphene reveals a reduction in the local density of states for bias voltages in the range of ±0.1 V.     

Comparison between sonodynamic effect and photodynamic effect with photosensitizers on free radical formation and cell killing

Although enhancement of ultrasound-induced cell killing by photodynamic reagents has been shown, the sonochemical mechanism in detail is still not clear. Here, comparison between sonodynamic effect and photodynamic effect with photosensitizers at a concentration of 10 μM on free radical formation and cell killing was made. When electron paramagnetic-resonance spectroscopy (EPR) was used to detect 2,2,6,6-tetramethyl-4-piperidone-N-oxyl (TAN) after photo-irradiation or sonication with 2,2,6,6-tetramethyl-4-piperidone (TMPD), the order of TAN formation in the photo-irradiated samples was as follows: rhodamine 6G (R6) > sulforhodamine B (SR) > hematoporphyrin (Hp) > rhodamine 123 (R123) > rose bengal (RB) > erythrosine B (Er) = 0; although there was time-dependent TAN formation when the samples were sonicated, no significant difference among these agents were observed. All these agents suppressed ultrasound-induced OH radical formation detected by EPR-spin trapping. Sensitizer-derived free radicals were markedly observed in SR, RB and Er, while trace level of radicals derived from R6 and R123 were observed. Enhancement of ultrasound-induced decrease of survival in human lymphoma U937 cells was observed at 1.5 W/cm² (less than inertial cavitation threshold) for R6, R123, SR and Er, and at 2.3 W/cm² for R6, R123, Er, RB and SR. On the other hand, photo-induced decrease of survival was observed for R6, Hp and RB at the same concentration (10 μM). These comparative results suggest that (1) ¹O₂ is not involved in the enhancement of ultrasound-induced loss of cell survival, (2) OH radicals and sensitizer-derived free radicals do not take part in the enhancement, and (3) the mechanism is mainly due to certain mechanical stress such as augmentation of physical disruption of cellular membrane by sensitizers in the close vicinity of cells and/or cavitation bubbles.     

Sonodynamic effects of protoporphyrin IX disodium salt on isolated sarcoma 180 cells

The cytotoxic effect of PPIX on isolated sarcoma 180 cells induced by ultrasound was investigated. Tumor cells suspended in air-saturated PBS (pH 7.2) were exposed to ultrasound at 2.2 MHz for up to 60 s in the presence and absence of protoporphyrin IX disodium salt (PPIX). The viability of cells was determined by a trypan blue exclusion test. The rate of ultrasonically induced cell damage was increased with 40–160 μM PPIX, while no cell damage was observed with 160 μM PPIX alone. This enhancement of cell damage with PPIX was inhibited by histidine. The participation of lipid peroxidation products in the cell damage process was also investigated. Scanning electron microscope (SEM) observation of the surface of cells was performed to evaluate the morphological changes induced by ultrasonic irradiation. The results indicate the involvement of a sonochemical mechanism.     

The geometric pattern of a pillared substrate influences the cell-process distribution and shapes of fibroblasts

Fibroblasts alter their shape, direction of movement, cytoskeleton arrangement, and focal contact when placed upon square array pillars. We prepared pillars of 1 μm diameter, separated by 3 μm, and having 1, 5, and 10 μm heights using substrates displaying identical surface chemistry. When cells seeded initially onto the tops of the pillars, fibroblasts subsequently were immobilized in situ by several pillars that visibly protruded through, but did not pierce, the cell bodies. The cytoplasma then migrated outward with long straight lamella along the interval of the pillars and formed several discrete attachment zones at their side walls – the value of their form index (FI) was as high as 35 – which altered the cellular shape entirely. Most of the cells interacted with the pillar substrate by spreading preferentially in a particular direction, but some of them had the ability to undergo coincident two-direction (x and y) migration; right-angle turn orientations led to the growth of dramatic cellular morphologies. Interestingly, this fibroblast's behavior variation was gradually in proportion to the pillar height of substrate. Our results confirm that cellular migration and cellular shape are both strongly affected by the geometry of the growth microenvironment.     

Structural properties of diamond fine particles and clusters prepared by detonation and decomposition of TNT

The thermal parameter B for three different particle sizes of diamond samples (bulk powder 1–4 μm, fine particle 144–195 Å and cluster 55–61 Å) was determined by the grazing incidence X-ray diffraction method. The values of B were found to be in the range 0.50–0.70 Å² for particles in the size range 195–55 Å and 0.27 Å² for 1–4 μm. All of them are larger than that of diamond bulk. A clear size dependence of B, increasing with decreasing particle size, was found. By analysing X-ray diffraction data at several temperatures the magnitude of B was found to be due to B_S (static part) instead of B_T (dynamic part). The average B_S values obtained were 0.04 Å², 0.19 Å² and 0.27 Å² for bulk powder, fine particle and cluster samples respectively. Ultrahigh resolution transmission electron microscope (TEM) observation confirmed the presence of strain, distortion, roughness and dislocation lines in many particles. TEM images of particles indicate that the clusters were not spherical in shape; they were mostly cubiform and some were truncated prism-like polyhedral. The present study reveals that the B_S component is responsible for the large B value in diamond fine particles and clusters. No clear surface local atomic distortion was found in the particles.     

Ultrasound exposure in the presence of hematoporphyrin induced loss of membrane integral proteins and inactivity of cell proliferation associated enzymes in sarcoma 180 cells in vitro

Ultrasonically induced effects of hematoporphyrin (HPD) on cell damage and membrane protein alteration of S180 isolated tumor cells in vitro were investigated, and the potential mechanisms of sonodynamic therapy (SDT) inhibiting tumor growth were discussed. Tumor cells suspended in air-saturated PBS (pH 7.2) were exposed to ultrasound at 1.8 MHz for up to 180 s in the presence and absence of HPD. The viability of cells was determined by a trypan blue exclusion test. To estimate the damage effects of SDT on plasma membrane of tumor cells primarily, membrane integral proteins (EGFR, Ras, Fas, FasL) and cell proliferation associated enzymes (adenylate cyclase and guanylate cyclase) were checked with immunochemical methods. The results indicated that the intensity threshold for ultrasonically induced cell damage at 1.8 MHz was 3 W/cm². At this condition, the expression of the integral proteins was obviously inhibited and the activity of the enzymes was decreased post ultrasound treatment in the presence of 20 μg/ml HPD. Loss of the membrane proteins and inactivity of AC and GC post SDT was time-dependent. This paper reveals SDT can cause the loss of tumor cell membrane integral proteins and inactivity of the enzymes associated with cell proliferation which might be attributed to a sonochemical activation mechanism. The mechanisms by that tumor growth is inhibited by SDT can be understood as that the growth signaling pathway is partially interdicted and the resistance of tumor cells to the specifically activated immune cells is weakened.     

Comparative study of morphology and surface composition of Al–Cr–Fe alloy powders produced by water and gas atomisation technologies

The morphology and surface composition of Al–Cr–Fe alloy powders of 0–63 and 63–100 μm size fractions, produced by gas and water atomisation, have been studied by scanning electron microscopy and Auger electron spectroscopy. While gas atomised particles are of spherical shape, water atomised powders are usually irregular in shape with a complex branched relief. The morphology and composition of surface oxides have been estimated. The surface oxide film is composed of aluminium oxides/hydroxides and contains no Fe and Cr atoms. Two to five water molecules are associated with one Al₂O₃ molecule on the surface of powders. The surface oxide film has a non-uniform thickness, with thick oxide islands separated by thinner oxide film. The parameters of the surface film morphology, such as the island coverage, the oxygen content and the thin film thickness, depend on the atomisation technology used and powder size fraction. Heavily and weakly oxidised powder groups present in all powder fractions are distinguished by Auger spectra analysis. Relationships between heavily and weakly oxidised powder groups are discussed as a function of atomisation technology and size fraction.     

Magnetoresistance in the magnetization reversal process of micropatterned Co wires

We measured the magnetoresistance (MR) and magnetization of Co wires of various widths in the range from 0.3 to 200 μm. The observed width dependence of some characteristics of MR is attributed to the change of the domain structure. As the width decreased below 1 μm, an abrupt jump appeared in the longitudinal MR. This can be interpreted as due to an abrupt reversal of the overall magnetization. The measured longitudinal MR was compared with the calculation in the Stoner–Wohlfarth model.     

A comparative study of donor formation in dysprosium, holmium, and erbium implanted silicon

Formation of donor centers in Czochralski grown silicon doped with dysprosium, holmium, and erbium is discussed. Donor states of three kinds are introduced in the implanted layers after annealing at T=700°C. Shallow donor states with ionization energies between 20 and 40 meV are attributed to oxygen -related thermal donors. Other donor centers in the energy range of E_C−(60…70) meV and E_C−(100…120) meV appear to be dependent on dopants. After a 900°C anneal strong changes in the donor formation are observed only in silicon doped with erbium. Instead of donors at E_C−(118±5) meV, new donor centres at E_C−(145±5) meV are formed. Reportedly, the latter ones are involved in the excitation process of the Er³⁺ ions with a characteristic luminescence line at ≈1.54 μm.     

NIR, MWIR and LWIR quantum well infrared photodetector using interband and intersubband transitions

This paper presents the design, fabrication and characterization of a QWIP photodetector capable of detecting simultaneously infrared radiation within near infrared (NIR), mid wavelength infrared (MWIR) and long wavelength infrared (LWIR). The NIR detection was achieved using interband transition while MWIR and LWIR were based on intersubband transition in the conduction band. The quantum well structure was designed using a computational tool developed to solve self-consistently the Schrödinger–Poisson equation with the help of the shooting method. Intersubband absorption in the sample was measured for the MWIR and LWIR using Fourier transform spectroscopy (FTIR) and the measured peak positions were found at 5.3 μm and 8.7 μm which agree well with the theoretical values obtained 5.0 μm and 9.0 μm for the two infrared bands which indicates the accuracy of the self-consistent model. The photodetectors were fabricated using a standard photolithography process with exposed middle contacts to allow separate bias and readout of signals from the three wavelength bands. The measured photoresponse gave three peaks at 0.84 μm, 5.0 μm and 8.5 μm wavelengths with approximately 0.5 A/W, 0.03 A/W and 0.13 A/W peak responsivities for NIR, MWIR and LWIR bands, respectively. This work demonstrates the possibility of detection of widely separated wavelength bands using interband and intersubband transitions in quantum wells.     

Field emission properties and surface structure of nickel containing amorphous carbon

Nickel containing amorphous carbon (a-C:Ni) films have been deposited by filtered cathodic vacuum arc (FCVA) technique by introducing pure nickel into the graphite target. The field electron emission property of a-C:Ni was improved when compared to that of pure tetrahedral amorphous carbon (ta-C) by FCVA. The emission threshold field of a-C:Ni film is about 5 V μm⁻¹, whilst the threshold field of the ta-C film is about 13 V μm⁻¹. Raman spectroscopy suggests that the sp² clusters in the carbon film increase both in size and number when Ni is introduced. However, the emission was found to degrade to threshold fields beyond 20 V μm⁻¹ after the a-C:Ni film was left in ambient for a week. This observation is attributed to surface absorption of oxygen on the a-C:Ni film, as determined by X-ray Photoelectron Spectroscopy.     

Multi-color tunneling quantum dot infrared photodetectors operating at room temperature

Quantum dot structures designed for multi-color infrared detection and high temperature (or room temperature) operation are demonstrated. A novel approach, tunneling quantum dot (T-QD), was successfully demonstrated with a detector that can be operated at room temperature due to the reduction of the dark current by blocking barriers incorporated into the structure. Photoexcited carriers are selectively collected from InGaAs quantum dots by resonant tunneling, while the dark current is blocked by AlGaAs/InGaAs tunneling barriers placed in the structure. A two-color tunneling-quantum dot infrared photodetector (T-QDIP) with photoresponse peaks at 6 μm and 17 μm operating at room temperature will be discussed. Furthermore, the idea can be used to develop terahertz T-QD detectors operating at high temperatures. Successful results obtained for a T-QDIP designed for THz operations are presented. Another approach, bi-layer quantum dot, uses two layers of InAs quantum dots (QDs) with different sizes separated by a thin GaAs layer. The detector response was observed at three distinct wavelengths in short-, mid-, and far-infrared regions (5.6, 8.0, and 23.0 μm). Based on theoretical calculations, photoluminescence and infrared spectral measurements, the 5.6 and 23.0 μm peaks are connected to the states in smaller QDs in the structure. The narrow peaks emphasize the uniform size distribution of QDs grown by molecular beam epitaxy. These detectors can be employed in numerous applications such as environmental monitoring, spectroscopy, medical diagnosis, battlefield-imaging, space astronomy applications, mine detection, and remote-sensing.     

High power, long pulse duration, single-frequency Nd:YVO4 master-oscillator power-amplifier

An efficient single-frequency Nd:YVO₄ master-oscillator power-amplifier is described. Gated pulses from a CW diode-pumped ring laser were amplified by three Nd:YVO₄ amplifiers. Pulses of 1mJ energy; 2 kW peak power and 1 μs duration were obtained at a repetition frequency of 10 kHz. Analogue shaping of the input pulses was used to control the output pulse shape and thus extend the useful range of pulse widths from the 100 ns to the μs regime. A simple mathematical expression was used to model the results which provided reasonable agreement with the experimental work. This system offers a promising pump source for a quasi-CW singly-resonant optical parametric oscillators.     

MBE grown type-II MWIR and LWIR superlattice photodiodes

We report on the status of GaSb/InAs type-II superlattice diodes grown and fabricated at the Jet Propulsion Laboratory designed for infrared absorption 2–5 μm and 8–12 μm bands. Recent LWIR devices have produced detectivities as high as 8 × 10¹⁰ Jones with a differential resistance–area product greater than 6 Ohm cm² at 80 K with a long wavelength cutoff of approximately 12 μm. The measured internal quantum efficiency of these front-side illuminated devices is close to 30% in the 10–11 μm range. MWIR devices have produced detectivities as high as 8 × 10¹³ Jones with a differential resistance–area product greater than 3 × 10⁷ Ohm cm² at 80 K with a long wavelength cutoff of approximately 3.7 μm. The measured internal quantum efficiency of these front-side illuminated MWIR devices is close to 40% in the 2–3 μm range at low temperature and increases to over 60% near room temperature.     

Development of IR-FEL facility for energy science in Kyoto University

A mid-infrared free electron laser (FEL) has been constructed for energy science in the Institute of Advanced Energy, Kyoto University. The FEL system consists of a compact S-band Linac and an undulator to generate 4–13 μm coherent mid-infrared radiations. The Linac consists of a 4.5 cell rf gun with a thermionic cathode and a 3-m traveling-wave-type accelerator tube fed by 10 MW and 20 MW rf power, respectively. We have succeeded to produce 40 MeV, 40 mA and 3 μs electron beams. Last December, the 9.2 μm spontaneous emission from the undulator generated by 29.5 MeV electron beams was observed for the first time. Further optimization parameters of both the electron beam and the optical cavity are being pursued for an FEL lasing in the near future.