Two-photon fluorescence real-time imaging on the development of early mouse embryo by stages

Mouse zygotes are widely used in developmental biology and transgenic animal research. Two-photon laser scanning microscopy is particularly useful in four-dimensional observation of big and thick biological samples, such as mouse embryo. The early mouse embryo development from zygote to 8-cell stage compaction was observed in real-time by stages using two-photon laser scanning microscopy in this paper. During our experiment, several scanning parameters were optimized in different development stages. The initial cleavages of mouse embryo, from the zygote to 2-cell, 2-cell to 4-cell, 4-cell to 8-cell, and the compaction at 8-cell stage were observed. During the first stage, localized intracellular calcium elevation along with the cleavage furrow and the asymmetric zygote cytokinesis was detected. The relation between the asymmetry and the location of the second polar body was investigated. The rotational cleavage of mouse embryo was also observed in the experiment. These results would be helpful to further research on mammalian embryonic development.     

Preimplantation embryotoxicity after mouse embryo exposition to reactive oxygen species.

Exposure of either gametes or embryos to conditions and/or factors that generate oxidative stress has been associated with impaired early embryogenesis. The effects of reactive oxygen species (ROS) on mouse preimplantation development, depending of the ROS-concentration and time of exposition, were studied. Two-cell embryos were incubated with 5, 10, 25 and 50 microM of hydrogen peroxide (H2O2) for 30 and 60 minutes of exposition and allowed to develop for 72 h to study the quality of development. The incubation with 50 microM H2O2 for 30 or 60 minutes, strongly inhibited the 2-cell embryo development as compared to the control (p < 0.001). Twenty-five microM H2O2 produced inhibition of blastocyst formation (p < 0.001) and 10 microM H2O2 significantly decreased the percentages of expanded and hatched blastocysts, which resulted morphologically altered (p < 0.05 and p < 0.01, respectively). The higher H2O2 concentrations were able to elicit necrotic morphology in the 2-cell arrested embryos, while 10 microM H2O2 induced moderate damage with the arrested embryos partially fragmented. In conclusion, important causes for defective preimplantation development and for early embryo losses may be due to oxidative stress because early mouse embryos exposed to ROS for short times arrested at the first cellular cycle (2-cell) and/or impaired embryo differentiation and morphogenesis, being these effects ROS-concentration-dependent.     

基于层次模型的协同产品开发影响因素研究

针对协同产品开发的协同深度和协同层次问题,首先建立一个网络化产品开发的协同层次模型。基于层次模型,从技术、组织和经营三个方面建立协同层次的影响因素集,分解出具体的影响因子。最后应用差距分析法对影响协同的关键因素进行分析,并给出具体的应用实例。基于层次模型的影响因素分析能较好地解决网络化产品开发协同层次的多样性和动态性问题,可通过量化分析识别影响协同的关键因素,从而提高网络化产品开发的综合效益。     

Study on affecting factors of collaborative product development based on collaboration hierarchy model

Aiming at the levels of collaborative degree in web-based product development, a collaboration hierarchy model of this product development is developed in this paper. Based on the model, the affecting factors on collaboration levels are analyzed systematically from many aspects, such as technology, organization and business. A gap analysis method is studied in detail, and is applied in a real project. The application shows that it can solve the diverse problems of collaborative product development effectively, and help enterprises find out the critical factors that affect the collaboration. Translated from Modern Manufacturing Engineering, 2006, (6): 1–3, 9 [译自: 现代制造工程]     

Brief Note : Improved <i>in vitro</i> embryo development of stenospermic grape by putrescine

The goal of this study was to determine the effect of putrescine, added to the culture medium, on the in vitro development of stenospermic grape (Vitis vinifera L) embryos. The cross breedings of Perlón x G.C88552 and Perlón x Argentina were used. 0 (control), 2 and 4 mM of putrescine were added to the immature seed’s culture medium. In Perlón x Argentina, 2mM of putrescine statistically increased the percentage of total embryos, direct germination, polyembryos and normal plants. In Perlón x G.C88552, only 2 mM of putrescine increased all the variables considered, eventually tripling the percentage of normal plants obtained. The results suggest that the endogenous concentration of putrescine may be a growth limiting factor. Adding putrescine to the culture medium of immature grape seeds is a legitimate resource to significantly increase the results of this technique.     

Tissue expression of platelet endothelial cell adhesion molecule-1 at pre and postnatal murine development.

Endothelial cells, at the cell-cell borders, express PECAM-1, and have been implicated in vascular functions. The monoclonal antibody MEC 13.3 recognizes PECAM-1 molecule from mouse vessels and allows to analyze the ontogeny of mouse endothelium. At the present, little is known about the molecular basis of differentiation pathways of endothelial cells, that enables its morphological heterogeneity. The purpose of this study was to analyze the pattern of PECAM-1 expression, employing monoclonal antibody MEC 13.3, in cellular suspensions obtained from different mouse organs at pre and postnatal stages. Fluorescence activated cell sorter analysis showed a different profile of the glycoprotein expression in a cell population with size and granularity selected by 1G11 endothelial cell line. The expression differs from prenatal to postnatal developmental stages in a given organ, and among the organs studied. Another cell population, with a size and granularity higher than IG11 endothelial cell line, coexists in cellular suspensions obtained from liver, gut and brain. These cells could be related to those detected by means of immunoenzyme methods which showed a non-differentiated morphology. The different PECAM-1 pattern expression could reflect potential organ-specific differentiation pathways during development and according to organs environment. The existence of another cell population with a size and granularity higher than 1G11 endothelial cell line required a phenotypic characterization.     

Quantitative tomography of early mouse embryos: laser scanning microscopy and 3D reconstruction

The cell volume alteration participates in a wide variety of cellular functions that may interfere with intra‐cellular homeostasis. The most adequate approach of estimation of the volume changes induced by osmotic misbalance, alteration in shape and size due to the action shape forming substances, etc., is the direct measurement of volumetric parameters of embryos. In the given research, the volume magnitude and kinetics of changes in volume and surface area of blastomere and polar bodies of early mouse embryos were determined using three‐dimensional reconstruction of the optical section stack obtained with laser scanning microscope (LSM). The size and surface area were determined for isotonic and anisosmotic conditions. The physiological significance of the findings is discussed.     

Ultrastructural observations of programmed cell death during metanephric development in mouse

Previous studies revealed apoptosis as an only programmed cell death (PCD) during renal morphogenesis before alternative type of PCD, necroptosis were introduced. Evidences of non‐apoptotic PCD during renal development were scarce and needed to be accumulated. The purpose of this study is to investigate whether non‐apoptotic PCD is involved in and observe ultrastructural features of apoptotic cells or non‐apoptotic PCD during metanephros development. For this purpose, light and transmission electron microscopy were used. The most significant finding to come out of this study was that necroptosis was observed during developing metanephros by electron microscopy. The results also provided another fact that apoptosis and necroptosis constituted the PCD during embryonic development of kidney in mouse. Compared to necroptosis, apoptosis was more predominantly evident throughout whole development period and in every compartment of metanephros except for proximal tubule. However, necroptosis was only exhibited in developing nephrons also except for proximal tubule. In addition, outcomes of PCD were related to morphogenetic features of metanephric development. Efferocytosis for apoptotic cell or bodies took place in each type cell and whole period of developing metanephros. Besides efferocytosis blood flow and urine flux were available to remove the corpses of PCD, especially PCD from developing nephrons. Our findings suggested that both apoptosis and necroptosis play important roles during nephrogenesis and observed three ways to clear the PCD cell: efferocytosis, blood flow, and urine flux. Microsc. Res. Tech. 76:467–475, 2013. © 2013 Wiley Periodicals, Inc.     

Relaxation of the mouse pubic symphysis during late pregnancy is not accompanied by the influx of granulocytes

In some animals, such as mice and guinea pigs, a hormonally controlled mechanism increases the flexibility of the pubic symphysis and enhances the cervical remodeling necessary for safe delivery. Cervical ripening during pregnancy is associated with a paradoxical influx of leukocytes. However, the changes in cell metabolism during relaxation of the mouse pubic symphysis for delivery have not been extensively studied. In this work, we used light microscopy and transmission and scanning electron microcopy, as well as immunohistochemistry and Western blotting for MMP-8, to investigate the involvement of granulocytes or resident stromal cells in the relaxation of the virgin pubic symphysis during late pregnancy (days 18 and 19, before delivery) in vivo and in explanted joints. MMP-8 was studied because this collagenase is a hallmark for cervical ripening associated with the influx of granulocytes during late pregnancy. Extensive dissolution and disorganization of the extracellular matrix was seen around fibroblastic-like cells in late pregnancy. In contrast to the cervix (positive control), morphological and immunohistochemical analyses revealed that there was no characteristic cellular inflammatory response in the interpubic tissue. Staining for MMP-8 was observed in chondroid and fibroblastic-like cells of virgin and relaxed interpubic ligament, respectively. However, no granulocytes were seen during the extensive remodeling of the pubic joint in late pregnancy. These results indicate that constitutive stromal cells may have an important role in tissue relaxation during remodeling of the pubic symphysis in pregnancy.     

光谱技术和仪器的新发展

从信息时代和高科技发展角度 ,综合评述了光谱分析检测技术和光谱仪器的最新发展态势 ,对在前沿科学技术发展成果基础上的若干光谱仪器的新发展方向也作了简略介绍。     

Measurements of the diameters of the great arteries and semi-lunar valves of chick and mouse embryos

The great arteries of embryos are small channels of a complex three-dimensional arrangement. Measurements of their diameters, as required for understanding cardiovascular morphogenesis and the genesis of malformations, cannot be performed in two-dimensional histological sections. We present and evaluate a quick and simple method for performing highly significant and objective measurements of the diameters of blood vessels in vertebrate embryos and used this method for providing statistics of the diameter of the semi-lunar valves and the lumina of the great arteries of early chick and mouse foetus. We employed the high-resolution episcopic microscopy technique for generating volume data and three-dimensional computer models of the arterial trees of 30 chick embryos (Hamburger Hamilton stage 34), 30 mouse embryos of the OF1 strain harvested on 14.5 dpc, 30 embryos of the OF1 strain harvested on 15.5 dpc and 28 mouse embryos of the PARKES strain harvested on 14.5 dpc. The three-dimensional models (voxel size 2 μm × 2 μm × 2 μm and 3 μm × 3 μm × 3 μm) were used for defining virtual resection planes perpendicular to the longitudinal axis of the blood vessels at comparable positions. In these planes, we measured the lumen areas and the lumen perimeters. We also calculated the lumen diameter and the true lumen area from the perimeter and present statistical analysis. Finally, we evaluate and discuss the reliability and reproducibility of our method and present all measurements in a form that minimizes the influence of specimen size variation, specimen processing and data generation methods.     

Imaging of mouse experimental melanoma in vivo and ex vivo by combination of confocal and nonlinear microscopy

We investigated possibilities of the combination of the one- and two-photon excitation microscopy for examination of the experimental melanoma tissue in vivo, in mice under general anesthesia, and ex vivo on freshly harvested specimens. Our aim was to obtain sufficiently informative images of unstained tumor tissues and their modifications after hyperthermia treatment. The mouse experimental melanoma structure was studied and compared with normal tissue from the same animal by using confocal and nonlinear microscopy techniques based on (i) one-photon excitation (1PE) fluorescence, (ii) 1PE reflectance, (iii) second harmonic generation imaging, and (iv) two-photon excitation autofluorescence. We checked different spectral conditions and other settings of image acquisition, as well as combinations of the above imaging modalities, to fully exploit the potential of these techniques in the evaluation of treated and untreated cancer tissue morphology. Our approach enabled to reveal the collagen fiber network in relation with the other tissues, and to identify invasive tumor cells. It also proved to be useful for the examination of interrelationships between functional and morphological aspects based on optical properties of the tissues, especially in studies of changes between the tumor and control tissue, as well as changes induced by physical treatments, e.g., delivery of microwave hyperthermia treatment. These differences were also evaluated quantitatively, when we found out that the maximum Euler–Poincaré characteristic reflects well the melanoma morphological structure. The results showed that the proposed investigative approach could be suitable also for a direct evaluation of tissue modifications induced by clinical interventions. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc.     

分析仪器的信息化发展趋势

分析仪器和其他传统工业一样,正在信息技术的改造下,向微型化、智能化、自动化和网络化方向发展。分析仪器作为数据获得的源头技术,必将在信息技术发展中起到重要作用,将从被动转向主动,并促进信息技术的发展。分析工作者也将是未来物联网不可或缺的生力军。     

Cloning and analysis of IFRG (interferon responsive gene) in rabbit oocytes and preimplantation embryos.

Although there is more evidence that shows that IFNs (interferons) plays a very important role in the early development of the embryo, the mechanism of IFNs is still unclear. Our study showed that IFRG is expressed from oocytes- through to the preimplantation embryo in rabbits. This finding provides some clues for better understanding the role of IFNs in the development of the embryo. The full length of rabbit IFRG cDNA (Accession No. AJ584672), with a 2794bp encoding 131 amino acid sequence, was cloned IFRG expression can be detected in 8 different tissues: ovary, heart, lung, liver, kidney, spleen, cerebra, and the 18-day whole-body embryo. Whole-mount in situ hybridization showed that IFRG was highly expressed in the inner-cell mass of rabbit blastula. IFRG may play an important role in embryo development and tissue differentiation.     

The structure of the developing chick retinal pigment epithelium revealed by high resolution scanning electron microscopy

The retinal pigment epithelium (RPE) in the developing eye of chick embryos has been studied during the early stages of development by high resolution scanning electron microscopy (HRSEM). Specimen preparation techniques which involve removal of the cytoplasmic matrix permitted visualization of organelles and other subcellular structures within RPE cells in detail and in three dimensional (3-D) stereo HRSEM. Using this technique, we were able to examine changes in melanosome structures during development and demonstrate that pigmentation in the RPE was present by day 4 of development. RPE plasma cell membranes showed extensive folding of the apical portion of the membrane closest to the developing neural retina by day 9. Examination of RPE photoreceptor junction revealed photoreceptor inner segments by day 6 and an outer segment by day 9. Mitochondria in the RPE were found to contain tubular cristae only. The ultrastructure in 3-D of the Golgi apparatus, smooth and rough endoplasmic reticulum, lysosomes and nuclear chromatin of the RPE, and Bruch's layer was revealed by the HRSEM method.     

Determination of Cliff-Lorimer k factors by analysis of crystallized microdroplets

The Cliff-Lorimer ratio technique for thin foils was used to determine a set of k factors at 120 kV for calibration of the energy dispersive X-ray analyser on a Philips EM400 analytical electron microscope. The standards used were the crystallized forms of microdroplets of solutions of inorganic salts. These experimental data were compared with other experimental work and found to agree within the experimental error with theoretical k factors.     

An easy brain thick-section-clearing protocol to observe antigens in the brains of neurological disease mouse models by conventional epifluorescence and confocal laser scanning microscopy

We developed a brain thick-section-clearing (BTS-C) procedure to observe coronal sections of the entire mouse brain with conventional epifluorescence microscopy (EFM). After clearing with the BTS procedure, the white light transmittance of the 1-mm-thick mouse brain sections was more than 96%. The distribution patterns of amyloid plaques or α-synuclein in 1-mm-thick brain sections of five different mouse strains cleared with this procedure were easily observed by EFM. In addition, the detailed distribution of antigens at higher magnifications was revealed by observing the same slides with a confocal laser scanning microscope (CLSM). In conclusion, we have shown that the BTS protocol can replace laborious and time-consuming semi-thin-sectioning procedures, and the cleared 1-mm-thick sections can be examined by EFM and CLSM to elucidate the distribution patterns of antigens.     

MSC.Patran二次开发及其集成开发环境

PCL语言是大型有限元分析软件MSC.Patran为用户二次开发提供的实用工具.对用PCL语言进行MSC.Patran二次开发的方法和流程进行了研究,编制了胶接修理参数分析系统.通过利用批处理命令调用MSC.Patran内置预处理程序的方法,将PCL开发环境与批处理程序集成,充分利用批处理命令的强大功能,使MSC.Patran的开发更加方便快捷.     

用.NET开发SolidWorks Add-in

分析.NET平台下非托管SolidW orks COM组件访问的技术原理,对该平台下SolidW orks COM对象向SolidW orksPIA对象模型转变的规则作了比较详细的归纳总结。并运用实例阐述在.NET环境下开发SolidW orks Add-in的实现过程与步骤,以期为在.NET平台下进行SolidW orks的二次开发与研究起到探索的作用。