Spindle abnormalities in normally developing and arrested human preimplantation embryos in vitro identified by confocal laser scanning microscopy

Hum. Reprod., 20, 672–682, 2005 The authors acknowledge     

Non-invasive measurement of pyruvate and glucose uptake and lactate production by single human preimplantation embryos

The consumption of pyruvate and glucose and the production of lactate by 40 single human preimplantation embryos has been measured using a non-invasive technique. Twelve of the embryos showed abnormal fertilization. Of the 28 normally fertilized embryos, nine (32%) developed to the blastocyst stage in culture while the remainder degenerated or arrested during cleavage. In the normal embryos, pyruvate uptake exceeded that of glucose in the early developmental stages (days 2-5 post-insemination) before glucose became the predominant substrate in the blastocyst (day 6). Considerable quantities of lactate were formed throughout development, rising from a value of 43.6 pmol/embryo/h on day 2.5 to 95.4 pmol/embryo/h on day 5.5. The values of pyruvate and glucose uptake and lactate production of those embryos which arrested were below those which developed normally. On the basis that one mole of glucose can give rise to two moles of lactate, only 50% of the lactate produced could be accounted for in terms of glucose uptake from the medium. This figure rose to 90% in the blastocyst. The remaining lactate must be derived from endogenous sources, most probably glycogen. It is proposed that the high production of lactate by human preimplantation embryos in vitro is an adaptation to the conditions of culture.     

Non-invasive measurement of glucose and pyrovate uptake by individual human oocytes and preimplantation embryos

Pyruvate and glucose uptake by 73 individual human oocytes andpreimplantation embryos was measured non-invasively, using anultramicrofluorescence assay to analyse changes in substratelevels in mkrodroplets of culture medium. The uptake of bothsubstrates was measured over successive daily incubations betweendays 1 (unfertilized oocytes) or 2 (‘spare’ embryoswhich were not transferred) and day 6 (day 0 = day of insemination).Under these conditions, 58% (25/43) of fertilized embryos withtwo pronuclei on day 1 developed to the blastocyst stage byday 6. The pyruvate uptake of these embryos increased from –28to a maximum of 40 pmol/ embryo/h between days 2.5 and 4.5.Similarly, glucose uptake increased from 8 to 14 pmol/embryo/hbetween days 2.5 and 4.5, but then increased further to 24 pmol/embryo/hon day 5 at the blastocyst stage. The pyruvate uptake of fertilizedembryos which arrested at cleavage stages was significantlylower than for those which developed to the blastocyst stage.Polyspermk and parthenogenetic embryos, and unfertilized oocytesalso had lower pyruvate uptakes at later stages. The glucoseuptake of unfertilized oocytes and abnormal embryos never reachedthe level of fertilized embryos at the blastocyst stage on day5.5. Non-invasive measurement of pyruvate uptake before embryotransfer may provide a valuable functional criterion for theselection of viable embryos capable of developing to the blastocyststage.     

激光共聚焦显微术在检测脑胶质瘤蛋白共表达中的应用

目的 了解ephrinB2及其受体EphB4蛋白在脑胶质瘤中的表达规律，并评价激光共聚焦显微术在检测脑胶质瘤组织和细胞蛋白共表达中的应用性。方法 利用双标免疫荧光分别检测EphB4/ephrinB2蛋白与GFAP或CD34蛋白在35例脑胶质瘤新鲜标本及人脑胶质瘤细胞系CHG-5、SHG-44中的共表达状况，以Leica SP2激光共聚焦显微镜观察、摄像并分析。结果 EphB4或ephrinB2蛋白与CD34蛋白可共表达于部分间质血管，EphB4和ephrinB2在肿瘤血管的表达主要定位于血管内皮细胞。在瘤组织内肿瘤细胞及两种细胞系，亦可见EphB4或ephrinB2蛋白与GFAP的共表达。SHG-44细胞中EphB4或ephrinB2红色荧光强度强于CHG-5细胞，而CHG-5细胞GFAP绿色荧光强度较之SHG-44细胞则明显增强。结论 (1)脑胶质瘤间质可能存在不同免疫表型的血管内皮，提示肿瘤血管的不同属性；(2)EphB4/ephrinB2蛋白表达可能与肿瘤细胞的分化程度有关；(3)双标免疫组化结合激光共聚焦显微术是一种可用于观测肿瘤蛋白共表达的良好方法。     

共聚焦激光扫描显微镜对厚样本光学断层及其三维重建

目的：探讨共聚焦系统各参数对厚样本光学断层、三维重建的影响。方法：用共聚焦系统对醛诱导后的大鼠主动脉作光学断层。分析激光强度、探测针孔直径、扫描步距等参数对内弹性膜三维重建的影响。结果和结论：(1)针孔为1Airy单位,调整激光强度以满足离开焦点1／2 Z-轴分辨率距离时,焦平面最强荧光信号减弱50％,此条件下获得的光学切片分辨率和信息完整性达最佳平衡,切片厚度可经验地定视为等于系统Z轴分辨率。(2)在上述条件下,扫描步距为Z-轴分辨率时,重建结构完整。(3)当扫描步距不变,针孔小于1Airy单位时,重建结构部分缺损;针孔大1Airy单位时,重建结构模糊。(4)样本经透明处理后,断层深度可增大4倍。     

The clustering and morphology of chondrocytes in normal and mildly degenerate human femoral head cartilage studied by confocal laser scanning microscopy

Chondrocytes are the major cell type present in hyaline cartilage and they play a crucial role in maintaining the mechanical resilience of the tissue through a balance of the synthesis and breakdown of extracellular matrix macromolecules. Histological assessment of cartilage suggests that articular chondrocytes in situ typically occur singly and demonstrate a rounded/elliptical morphology. However, there are suggestions that their grouping and fine shape is more complex and that these change with cartilage degeneration as occurs in osteoarthritis. In the present study we have used confocal laser scanning microscopy and fluorescently labelled in situ human chondrocytes and advanced imaging software to visualise chondrocyte clustering and detailed morphology within grade‐0 (non‐degenerate) and grade‐1 (mildly degenerate) cartilage from human femoral heads. Graded human cartilage explants were incubated with 5‐chloromethylfluorescein diacetate and propidium iodide to identify the morphology and viability, respectively, of in situ chondrocytes within superficial, mid‐ and deep zones. In grade‐0 cartilage, the analysis of confocal microscope images showed that although the majority of chondrocytes were single and morphologically normal, clusters (i.e. three or more chondrocytes within the enclosed lacunar space) were occasionally observed in the superficial zone, and 15–25% of the cell population exhibited at least one cytoplasmic process of ~ 5 μm in length. With degeneration, cluster number increased (~ 50%) but not significantly; however, the number of cells/cluster (P < 0.001) and the percentage of cells forming clusters increased (P = 0.0013). In the superficial zone but not the mid‐ or deep zones, the volume of clusters and average volume of chondrocytes in clusters increased (P < 0.001 and P < 0.05, respectively). The percentage of chondrocytes with processes, the number of processes/cell and the length of processes/cell increased in the superficial zone of grade‐1 cartilage (P = 0.0098, P = 0.02 and P < 0.001, respectively). Processes were categorised based on length (L0 – no cytoplasmic processes; L1 < 5 μm; 5 < L2 ≤ 10 μm; 10 < L3 ≤ 15 μm; L4 > 15 μm). With cartilage degeneration, for chondrocytes in all zones, there was a significant decrease (P = 0.015) in the percentage of chondrocytes with ‘normal’ morphology (i.e. L0), with no change in the percentage of cells with L1 processes; however, there were significant increases in the other categories. In grade‐0 cartilage, chondrocyte clustering and morphological abnormalities occurred and with degeneration these were exacerbated, particularly in the superficial zone. Chondrocyte clustering and abnormal morphology are associated with aberrant matrix metabolism, suggesting that these early changes to chondrocyte properties may be associated with cartilage degeneration.     

Concomitant use of Congo red staining and confocal laser scanning microscopy to detect amyloidosis in oral biopsy: A clinicopathological study of 16 patients

Twenty oral biopsies from 16 patients were analyzed both by traditional microscopy and by confocal laser scanning microscopy. Using conventional histopathological techniques, the diagnosis of amyloidosis was confirmed only in 15 biopsies. Using confocal laser scanning microscopy, amyloid deposits were detected in all of the samples. The current study shows that confocal laser scanning analysis helps to identify minimal amyloid deposits that could be overlooked using traditional microscopy, thus raising the sensitivity of oral biopsy up to 100%.     

低氧缺血海马神经元内Ca~(2 )动态变化的LSCM测定

测定脑组织细胞内 Ｃａ~(2 )浓度是评价缺血引起神经元损伤的重要指标。本研究用激光扫描共聚焦显微镜（ＬＳＣＭ）和Ｆｌｕｏ－３／ＡＭ荧光探针标记技术,从单个活细胞水平检测缺血刺激诱发的海马神经元内Ｃａ２＋瞬间动态变化。结果显示低氧使胞内Ｃａ２＋浓度显著升高。谷氨酸引起Ｃａ２＋浓度升高缓慢但持续时间长。缺血对Ｃａ２＋浓度影响不显著。撤除葡萄糖则引起胞内Ｃａ２＋迅速降低。实验结果表明不同缺血刺激因素引起的钙振荡各异。用ＬＳＣＭ对活细胞内部非侵入光学断层扫描,能清晰记录到这些瞬态钙值变化。     

Proliferation of blastomeres from biopsied cleavage stage human embryos in vitro: an alternative to blastocyst biopsy for preimplantation diagnosis

Normally fertilized human embryos were biopsied at cleavagestages on the third day after in-vitro fertilization (IVF).One or two blastomeres at the 8-cell stage were removed andco-cultured with the biopsied embryos. Embryos and blastomereswere assessed daily for morphological development until day6, when the number of cells were counted by labelling the nuclei.In all, 53% of the biopsied embryos (25 out of 47) reached theblastocyst stage between day 5 and 6 and the proportion wasthe same irrespective of the number of cells removed. Therewas no significant difference between biopsied embryos fromwhich one or two blastomeres respectively had been removed withregard to total cell numbers at the blastocyst stage (56.2 ±3.0 and 64.7 ± 5.5), number of trophectoderm (45.4 ±3.5 and 44.0 ± 5.7) and inner cell mass cells (14.0 ±1.2 and 16.6 ± 1.8). Overall, 72% of the isolated blastomeresdivided at least once over 3 days in culture and 50% dividedmore than once. The mean overall cell number after 3 days inculture was 3.7 ± 0.48 per blastomere (range 1–8cells) if one cell was removed and 6.9 ± 1.0 if two cellswere removed. If the undivided blastomeres are excluded, themean cell number was 4.8 ± 0.51 and 8.3 ± 1.0respectively. Over this period, 55% of the blastomeres cavitated.Of the blastomeres taken from embryos that developed to theblastocyst stage, 92% divided and 76% cavitated. In those fromarrested embryos, 50% divided (P < 0.002) and 32% cavitated(P < 0.003). From the first group 8% of blastomeres and fromthe second group 41% of blastomeres neither divided nor cavitated(P < 0.008). We conclude that as a more consistent alternativeto blastocyst biopsy, cryopreserving biopsied cleavage stageembryos and culturing blastomeres would increase the numberof cells available for genetic analysis. This could facilitatepreimplantation diagnosis of inherited disease by improvingreliability and possibly allowing combined detection of chromosomaland single cell defects. Further studies will investigate thegenetics of proliferated blastomeres.     

Human preimplantation development in vitro is not adversely affected by biopsy at the 8-cell stage

Normally fertilized human embryos biopsied 3 days after in-vitro fertilization (IVF) have been examined for effects on viability and development in vitro after removal of one or two cells at the 8-cell stage (1/8 and 2/8) from each embryo. A high proportion of 7/8 and 6/8 biopsied and unmanipulated embryos developed to the blastocyst stage between days 5 and 6 (79, 71 and 59%, respectively), and many biopsied embryos (56%) hatched from the zona pellucida in vitro. The viability of biopsied embryos which developed to the blastocyst stage was assessed by daily non-invasive measurement of the uptake of two energy substrates, glucose and pyruvate. Uptake of both substrates was generally lower in 7/8 and 6/8 biopsied embryos but only in proportion to the reduced cellular mass. The total cell number and the numbers of both trophectoderm (TE) and inner cell mass (ICM) cells in biopsied embryos at the blastocyst stage, counted by differential labelling of their nuclei, were also reduced in proportion but the ratio of ICM to TE cells was maintained in both 7/8 and 6/8 biopsied embryos. We conclude that removal of one or two cells at the 8-cell stage, while reducing the cellular mass, does not adversely affect the preimplantation/development of biopsied embryos in vitro and suggest that this approach could be used for preimplantation diagnosis of genetic defects.     

Imaging early stages of the female reproductive structure of arabidopsis by confocal laser scanning microscopy

Background : The gynoecium is the female reproductive structure and probably the most complex plant structure. During its development, different internal tissues and structures are formed. Insights in gene expression or hormone localization patterns are key to understanding gynoecium development from a molecular biology point of view. Results : Imaging with a confocal laser scanning microscope (CLSM) is a widely used strategy; however, visualization of internal developmental expression patterns in the Arabidopsis gynoecium can be technically challenging. Here, we present a detailed protocol that allows the visualization of internal expression patterns at high resolution during gynoecium development. We demonstrate the applicability using a cytokinin response marker (TCS::GFP), an auxin response marker (DR5::VENUS), and a SEPALLATA3 marker (SEP3::SEP3:GFP). Conclusions : The detailed protocol presented here allows the visualization of fluorescence signals in internal structures during Arabidopsis gynoecium development. This protocol may also be adapted for imaging other challenging plant structures or organs. Developmental Dynamics 244:1286–1290, 2015. © 2015 Wiley Periodicals, Inc.     

Confocal laser scanning microscopy and three-dimensional reconstruction of cell clusters in serous fluids

Thick cell clusters are a common finding in reactive and malignant effusions. In order to arrive at a diagnosis, clusters are evaluated for certain cytomorphologic features including size, shape, smooth vs. scalloped borders, and three-dimensional (3-D) configuration. By conventional microscopy, the image of these clusters is often blurred due to limitations in resolution. Consequently, the exact internal structure and cellular arrangement within these clusters cannot be adequately determined. Utilizing confocal laser scanning microscopy (CLSM), we examined serous fluids from a variety of conditions. Cases included mesothelioma, adenocarcinoma, and papillary adenocarcinoma. Smears were stained with 0.01% ethidium bromide and 1% eosin Y, followed by analysis with an ACAS 570™ image analyzer (Meridian Instruments, Inc. Okemos, MI). Serial confocal fluorescence images were acquired, which allowed 3-D reconstruction of the clusters. Mesothelioma clusters (excluding those with obvious central collagen cores by light microscopy) appeared to be formed of the following configurations: 1) randomly coiled cords of cells, 2) small papillae encompassing central cores, and 3) tissue fragments with pseudoacinar formation. In contrast, adenocarcinomas had a more orderly pattern, with tightly cohesive cells and true acinar formation. Diagn. Cytopathol. 1997;17: 272–279. © 1997 Wiley-Liss, Inc.     

An investigation by mathematical modelling of whether mouse and human preimplantation embryos in static culture can satisfy their demands for oxygen by diffusion

Mammalian preimplantation embryos are conventionally grown in small, static droplets of medium. By contrast, embryos within the oviduct are subject to mixing forces arising from the action of cilia and the contractions of the myosalpinx. Such forces will minimize the buildup of unstirred layers around the embryos and facilitate the exchange of gases and metabolites. We have devised a mathematical model to investigate whether preimplantation mouse and human embryos grown in static culture can satisfy their requirement for oxygen solely by diffusion i.e. in the absence of stirring. The model incorporates the diffusion coefficients for oxygen in the medium outside and within the embryo, the oxygen tension of the culture medium, the size of the embryo and its oxygen uptake. Solutions of the model are provided for mouse oocytes and 2-cell embryos, mouse blastocysts and human morulae. In each case, the model is solved for two different values of the diffusion coefficient of oxygen within the cell and of the oxygen content of the medium (5 and 20%). The conclusion is that mouse embryos in static culture are likely to be able to satisfy their demands for oxygen by diffusion alone, but that human embryos may become marginally hypoxic, especially at lower oxygen levels.     

Perivascular structures in corrosion casts of the human central nervous system: A confocal laser and scanning electron microscope study

Scanning electron microscopy (SEM) of microvascular corrosion casts revealed perivascular structures that resembled smooth muscle and pericyte cells. Although these structures have been studied in widely different experimental contexts, their origin, function, and distribution pattern in different tissues are not understood. Microvascular corrosion casts from 15 fresh human brains and 20 lumbar spinal cords were studied by SEM. In five cerebral hemispheres a fluorescent resin was injected in order to study the vascular bed by confocal laser scanning microscopy (CLSM). Microvascular casts showed two perivascular structures on their surfaces: plastic strips, which formed a muff around arteriolar vessels, and pericyte-like structures that were present around the capillary network. Their morphological characteristics and distribution were similar to those of smooth muscle cells and pericytes, respectively. The SEM study showed that these structures were not tightly joined to the cast surface, but were connected to the vascular cast by narrow plastic connections. The CLSM showed that the resin invaded the subendothelial space, thus giving rise to these structures. Perivascular structures associated with arteriolar and capillary vessels appear to represent smooth muscle cells and pericytes. They are formed by the passage of the resin to the subendothelial space, probably through weak endothelial cell junctions. The effusion of resin into the subendothelial space may represent evidence for the structural basis of myocyte and pericyte cell control. Chemical communication by substances released locally or transported to these cells through these junctions may regulate their functions, allowing them to regulate blood flow. Anat. Rec. 252:176–184, 1998. © 1998 Wiley-Liss, Inc.     

大鼠海马神经干细胞体外增殖、凋亡和分化的激光扫描共焦显微镜观察

目的 采用激光扫描共焦显微镜观察体外培养胎鼠海马神经干细胞（NSCs）增殖、凋亡及其分化情况。方法 体外分离培养胚胎大鼠海马NSCs,把神经干细胞球培养在共焦显微镜专用培养皿中。用 Hoechst 33258染细胞核,免疫荧光细胞化学技术检测巢蛋白（Nestin）的表达以鉴定NSCs,检测神经元、星形胶质细胞的特异性标记物β-Ⅲ型微管蛋白（β-Ⅲ tubulin）和胶质纤维酸性蛋白（GFAP）的表达以测定NSCs的分化能力。通过激光扫描共焦显微镜对神经干细胞球进行光学连续断层扫描,然后用软件进行三维重建,立体动态观察神经球。结果 通过激光扫描共焦显微镜观察到神经球     

Time from insemination to first cleavage predicts developmental competence of human preimplantation embryos in vitro

BACKGROUND: The absence of reliable markers for the identification of viable embryos for transfer at the early cleavage stage is likely to contribute to the generally low implantation rates and high incidence of multiple gestation in IVF treatment. In this study, we investigate the relationship between timing of first cleavage and the incidence of blastocyst formation in vitro. METHODS: Couples (n = 70) with at least one embryo remaining after transfer were included in the analyses. All embryos (n = 579) were examined for early cleavage at 25 h after insemination. Following embryo transfer, the remaining embryos (n = 426) were cultured until day 7 of development, and assessed for blastocyst formation. RESULTS: Eighty-five embryos (14.7%) cleaved to the 2-cell stage within 25 h of insemination; 26 of these were selected for transfer on day 2. Of the 59 embryos remaining in culture, 19 (32.2%) developed to the blastocyst stage; this was a significantly higher number than was observed in embryos (61/367; 16.6%) that failed to cleave within 25 h of insemination (P < 0.01). Within these two groups of embryos the proportion of hatched blastocysts was 11/59 (18.6%) and 26/367 (7.1%) respectively (P < 0.005). CONCLUSIONS: These findings indicate that early cleavage is indicative of increased developmental potential in human embryos and may be useful as an additional criterion in the selection of embryos for transfer.     

Immunohistochemical analysis of neuroendocrine (NE) differentiation in testicular germ cell tumors (GCTs): use of confocal laser scanning microscopy (CLSM) to demonstrate direct NE differentiation from GCTs

Neuroendocrine (NE) differentiation is infrequent in testicular tumors and its histogenesis is not well understood. The present study is aimed at elucidating the pathway of neuroendocrine differentiation in germ cell tumors (GCTs) of the testis. In the analysis of 46 germ cell tumor components from 23 testicular tumors, we focused on GCTs with neuroendocrine differentiation, 7 teratoma, 1 embryonal carcinoma and 1 neuroendocrine carcinoma by immunohistochemical study and confocal laser scanning microscopy (CLSM) analysis. NE marker positive cells were noted in the tumor with collision of teratoma and embryonal carcinoma (E&T tumor), in the immature columnar cells of transitional form of embryonal carcinoma to teratoma (E-T cells) and neuroendocrine carcinoma cells, in addition to the well known mature intestinal mucosa in teratoma. Double staining for a NE marker (CGA) and a germ cell marker (PLAP) demonstrated the localization of both proteins in the same E-T cells confirmed by CLSM. Another finding, indicating the intimate relation between embryonal carcinoma and neuroendocrine differentiation, is that neuroendocrine carcinoma expressed a marker of embryonal carcinoma, CD30. The present results indicated that the NE cells might be differentiated from embryonal carcinoma, a view that has not been proposed before, but that is made in the present study using CLSM.     

Plastic casts and confocal laser scanning microscopy applied to the observation of enamel tubules in the red Kangaroo (Macropus rufus)

Scanning electron microscopy for plastic casts and confocal laser scanning microscopy for Villanueva bone-stained ground sections were used together to observe enamel tubules in red kangaroo molars. Although the tubular structures such as terminals, bends, expansions, splits, divergences and rejoinings in this species were within the variations of marsupial species, their morphological characteristics were demonstrated with extremely clear and persuasive images. Thus, the combined observations of plastic casts by scanning electron microscopy and Villanueva bone-stain sections by confocal laser scanning microscopy were found to be of value for the investigation of enamel tubules and tubular structures in other hard tissues.