Atmospheric plasma jet device for versatile electron microscope grid treatment

Atmospheric pressure plasmas have been widely applied in surface modification and biomedical treatment due to its ability to generate highly reactive radicals and charged particles. In cryogenic electron microscopy (cryo-EM), plasmas have been used in eliminating the surface contaminants as well as generating the hydrophilic surface to embed the specimen on grids. Particularly, plasma treatment is a prerequisite for negative stain and quantifoil grids,which are coated with hydrophobic carbon on the grid surface. Here we introduce a nonthermal atmospheric plasma jet system as an alternative new tool for surface treatment. Unlike the conventional glow discharger, we found that the plasma jet system successfully cleans the grid surface and introduces hydrophilicity on grids in the ambient environment without introducing a vacuum. Therefore, we anticipate the plasma jet system will be beneficial in many aspects, such as cost-effective, convenient, versatile, and potential applications in surface modification for both negative stain and cryo-EM grid treatment.

Image Compression in Morphometry Studies Requiring 21 CFR Part 11 Compliance: Procedure Is Key with TIFFs and Various JPEG Compression Strengths

This study aims to compare the integrity and reproducibility of measurements created from uncompressed and compressed digital images in order to implement compliance with 21 CFR Part 11 for image analysis studies executed using 21 CFR Part 58 compliant capture systems. Images of a 400-mesh electron microscope grid and H&E stained rat liver tissue were captured on an upright microscope with digital camera using commercially available analysis software. Digital images were stored as either uncompressed TIFFs or in one of five different levels of JPEG compression. The grid images were analyzed with automatic detection of bright objects while the liver images were segmented using color cube-based morphometry techniques, respectively, using commercially-available image analysis software? When comparing the feature-extracted measurements from the TIFF uncompressed to the JPEG compressed images, the data suggest that JPEG compression does not alter the accuracy or reliability to reproduce individual data point measurements in all but the highest compression levels. There is, however, discordance if the initial measure was obtained with a TIFF format and subsequently saved as one of the JPEG levels, suggesting that the use of compression must precede feature extraction. It is a common practice in software packages to work with TIFF uncompressed images. However, this study suggests that the use of JPEG compression as part of the analysis work flow was an acceptable practice for these images and features. Investigators applying image file compression to other organ images will need to validate the utility of image compression in their work flow. A procedure to digitally acquire and JPEG compress images prior to image analysis has the potential to reduce file archiving demands without compromising reproducibility of data.

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.

Shyntesis and Characterization of Quantum Dot Superlattices

The engineering of a new generation of advanced materials based on nanoparticles demands the fabrication of self-assembled arrays of passivated metal, oxide and semiconductor clusters (1 -). In particular the case of self-assembled gold clusters passivated with an organic molecule has attracted the attention of several researchers (2-3). Because its unique properties gold metal is a leading candidate for the fabrication of single electron tunneling devices.Passivated gold nanoclusters were produced using the method developed by Brust et.al. (4) with the modifications of Whetten et.al (5) n-alkylthiol molecules were used as passivating agent. Carbon chains from C=4 to C=18 were used. It was found that C=12 dodecanethiol was optimum for forming ordered arrays of the clusters. To produce the superlattice crystallization a toluene vapor atmosphere was used. The passivated clusters were deposited on a copper electron microscope grid covered with carbon.

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.