82COMPARISON OF THE SURVIVAL OF IN VITRO-DERIVED PORCINE OOCYTES AND EMBRYOS VITRIFIED BY OPEN PULLED STRAW, ELECTRON MICROSCOPE GRID, AND NYLON LOOP SYSTEM

Various sample containers have been developed for ultra-rapid vitrification of oocytes and embryos to minimize chilling and other injuries. In this study, the survival rates of in vitro-derived porcine oocytes were compared after cryopreservation using open pulled straw (OPS), electron microscope grid (EMG), and nylon loop system (NLS). In addition, the post-thaw survival of porcine morulae and blastocysts was assessed after vitrification by the OPS method. In vitro maturation and fertilization were performed according to the procedures of Funahashi et al., 1994 Theriogenology 41, 1425–1433). Fertilized oocytes were cultured in glucose-free NCSU 23 supplemented with 5mM sodium pyruvate, 0.5mM sodium lactate and 4mgmL−1 bovine serum albumin for 2 days at 39°C; 10% fetal bovine serum was added to the culture medium thereafter. Oocytes and embryos were treated with 7.5μgmL−1 cytochalasin B for 30min, centrifuged at 13,000g for 13min and then exposed sequentially to an ethylene glycol (EG) vitrification solution (0M for 1min, 2M for 5min and 8M plus 7% polyvinylpyrrolidone for 1min). Oocytes (n=100per treatment group) were placed in or on sample containers and plunged into liquid nitrogen. Porcine morulae and blastocysts (n=137) were similarly treated, aspirated into OPSs, and plunged into liquid nitrogen. Oocytes and embryos were thawed rapidly by transferring the sample container into 1M EG for 2min, and then the embryos were serially diluted by transfer into 0.5M for 2min, and medium for 5min. Post-thaw survival of vitrified oocytes was assessed by development after IVF and culture to morulae or blastocysts. Post-thaw survival of vitrified morulae and blastocysts was assessed by their ability to continue development to, respectively, blastocysts and expanded blastocysts. After oocytes were thawed, fertilized and cultured in vitro, the rates of development to the morula- or blastocyst-stage were 8%, 0% and 6% for, respectively, the OPS, EMG and NLS groups. The rate of development of non-cryopreserved control oocytes was 38% (38/100). Although significantly fewer oocytes developed after vitrification compared to the control (P<0.05), OPS and NLS containers were superior to EMG containers (P<0.05). Morula- or blastocyst-stage embryos vitrified using OPS yielded a significantly higher rate of survival than did oocytes (48%, 66/137; P<0.05). This study indicates that very few in vitro-matured porcine oocytes survive vitrification using OPS, EMG and NLS methods. Considerably higher rates of survival were obtained by in vitro-produced porcine morulae and blastocysts following vitrification by the OPS method. Additional research is needed to identify and develop methods to overcome the factors limiting the cryopreservation of porcine oocytes for practical uses. Supported by KOSEF (R05-2002-000-00388-0) grant.

Characterization of colloidal materials by cryo-TEM

Many researchers develop products and processes that depend upon colloidal materials. Rheological and other important material parameters are influenced by the morphology of the colloid. The photography industry uses many such colloids including silver halide sols, photographic coupler sols and emulsions surfactants, and gelatin solutions and gels. High-resolution direct electron images of these systems in their native states permit photographic scientists to understand how changes in formulation produce changes in microstructure and, ultimately, to understand the photographic performance of the system.We prepare thin biconcave liquid films spanning the holes in a perforated carbon film supported by an electron microscope grid (see Fig 1). To prevent changes in composition of the colloid caused by evaporation, the films are prepared in an environmental chamber and are propelled through a trap door into a container of liquid ethane. The specimen is cooled at a rate sufficient to prevent crystallization of water. When maintained below -150 °C, these specimens are stable and compatible with the high vacuuary of the electron microscope.

Growth and morphology of NaCl-type crystals prepared by various methods

NaCl type crystals generally grow a cube. NaCl-type crystals with rhombic prism shape have been found among the cube crystals. No convincing interpretation for the rhombic prism crystals has been given. The growth mechanisms of some crystals have been clarified by means of potetial energy calculations in previous papers. In the present paper, free energy of NaCl type crystals having various shapes have been calculated in order to make clear the origin of the appearing of the rhombic prism crystals. The growth mechanism is discussed on the basis of the free energy.NaCl crystals were grown from aqueous solution in a glass made growth cell thermostated with circulating water(±l⋅C). The NaCl crystals were grown at temperature of 50°C which is equivalent to the supersaturation of 2%. The grown crystals were mounted on a net and were dried with a filter paper as quickly as possible. The specimens were examined by a microscope. PbS crystals are prepared by the two-heater method which have been developed to produce ultrafine particles of low melting materials. The detail of the preparation has been shown in a previous paper. The grown crystals collected on a carbon film supported by an electron microscope grid were observed with a Hitachi H-9000 electron microscope.

TEM microstructure of laser-trimmed Si-Cr devices

Laser ablation adjustment of resistance values of resistors Incorporated within integrated circuits is common practice. We analyzed cross-section and plan-view Si-Cr laser ablated resistors in actual devices. Resistors were prepared as follows: nominally 500Å Si-Cr films were deposited on 2nd level quartz, patterned and then covered with 3rd level quartz. Oxygen was bled into the sputtering chamber during the Si-Cr deposition. No special heat-treatment was given to the Si-Cr resistors, but they did see 5-hours at 450C during the 3rd level quartz sputter. The resistors were contacted by 2-micron thick 2nd level Al-Cu metal stripes. The resistors were trimmed by focussed laser light impinging on the Si-Cr layer buried in SiO2. Trimming was accomplished, step-wise, using a YAG laser operating at different power settings. Plan-view samples were made by mechanically polishing to just above the resistor, inverting the sample, and mechanically polishing from the substrate side to just below the resistor. A copper microscope grid was epoxied on the thin specimen. After the two mechanical polishing operations, the sample was about one micron thick. Final thinning of the sample to about 0.5 μm was accomplished via a short ion-milling operation. Cross-sectioned samples are prepared the same way, taking care to have the final mechanical polishing operation terminate in the laser ablated area. No acids are used in either case. The samples see only acetone and water.

Phase transformations and kinetics in small particles

Small particles of metals supported on various substrates are important heterogeneous catalysts. The rate of catalysis depends critically on the surface morphology of these catalysts and can vary significantly due to strong metal substrate interactions. On the other hand the structure of an isolated small particle is ill-defined and has been shown to fluctuate between various forms, existing in a quasi-molten state. We present here results which shows the transformation of a small particle sitting in a deep potential well on a substrate with a stable shape, into a free floating state which is structurally unstable.The experimental procedure consisted of dispersing gold clusters produced by the reduction of organometallic compounds, onto magnesium oxide smoke particles which were collected onto a microscope grid placed over burning magnesium in air. The specimens were then imaged in a H- 9000 high resolution electron microscope operating at an accelerating voltage of 300 KV. The distribution of small gold panicles on the low energy (100) face of the MgO cube particles is shown in Fig.1