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

BACKGROUND: Despite recent technical improvements, many human preimplantation embryos fail to develop to the blastocyst stage or implant after transfer to the uterus. A possible cause for this developmental arrest is the high incidence of nuclear and postzygotic chromosomal abnormalities observed during cleavage, including chaotic chromosome complements, suggestive of defects in mitotic chromosomal segregation. The underlying mechanisms are largely unknown, but similarities with chromosome instability in human cancers led to the proposal that cell cycle checkpoints may not operate at these early stages. METHODS: To investigate this and to examine whether spindle abnormalities contribute to chromosome malsegregation, we have used fluorescence and confocal laser scanning microscopy, following immunolabelling with antibodies specific for alpha-tubulin, gamma-tubulin, or acetylated tubulin, combined with a DNA fluorochrome to visualize nuclei, spindle and chromosome configurations in normal and arrested human embryos, from cleavage to blastocyst stages. RESULTS: In addition to frequent interphase nuclear abnormalities, we identify for the first time various spindle abnormalities including abnormal shape and chromosome loss and multipolar spindles at cleavage and blastocyst stages. CONCLUSIONS: We propose that a major pathway leading to postzygotic chromosomal abnormalities is the formation of binucleate blastomeres with two centrosomes which result either in a bipolar spindle and division to two tetraploid blastomeres, or in a multipolar spindle, chromosome malsegregation and chromosomal chaos.     

Analysis of collagen distribution in human crown dentin by confocal laser scanning microscopy

The authors used confocal laser scanning microscope to analyze human crown dentin. Specimens from 10 teeth were divided in two groups, one of which was decalcified and stained with hematoxylin and eosin. In the second group an undecalcified section was analyzed. Both groups were scanned by confocal microscope to generate optically sectioned images. All of the analyzed samples presented an intense autofluorescent that was ascribed to collagens. The degree of autofluorescence intensity was variable and might be due to collagen expression. The results indicate that a confocal microscope may be of help in analyzing and defining the nature and extent of collagen fibrils in human dentin.     

Mitochondria in tumor cells studied by laser scanning confocal microscopy

We present here a confocal fluorescence microscopy study of mitochondria in sensitive and resistant carcinoma cells by using two potentiometric probes of mitochondria, rhodamine 123 (R123) and dimethylaminostyryl-methylpyridiniumiodine. We have found that active mitochondria in sensitive MCF-7 and multidrug resistant MCF-7/DX carcinoma cells are very different in localization and morphology. In sensitive cells active mitochondria are found in the perinuclear region, whereas in the multidrug resistance (MDR) subline they are confined to the cell periphery. Interestingly, the MDR revertant verapamil has been found to restore in MCF-7/DX cells the same pattern of active mitochondria seen in sensitive cells. We have also studied R123 in human lung carcinoma A549 cells, which display a low responsivity to doxorubicin, and overexpress the lung resistance-related protein. In addition to perinuclear mitochondria, peripheral mitochondria with weaker fluorescence can be seen in this cell line. Interestingly, in the two examined carcinoma lines we have been able to recognize by image analysis a common new star-lobed morphology. Our results indicate that in resistant carcinoma cells two populations of mitochondria coexist with different localization, morphology, and activity.     

Derivation of human embryonic stem cells from developing and arrested embryos

Human embryonic stem cells (hESC) hold huge promise in modern regenerative medicine, drug discovery, and as a model for studying early human development. However, usage of embryos and derivation of hESC for research and potential medical application has resulted in polarized ethical debates since the process involves destruction of viable developing human embryos. Here we describe that not only developing embryos (morulae and blastocysts) of both good and poor quality but also arrested embryos could be used for the derivation of hESC. Analysis of arrested embryos demonstrated that these embryos express pluripotency marker genes such OCT4, NANOG, and REX1. Derived hESC lines also expressed specific pluripotency markers (TRA-1-60, TRA-1-81, SSEA4, alkaline phosphatase, OCT4, NANOG, TERT, and REX1) and differentiated under in vitro and in vivo conditions into derivates of all three germ layers. All of the new lines, including lines derived from late arrested embryos, have normal karyotypes. These results demonstrate that arrested embryos are additional valuable resources to surplus and donated developing embryos and should be used to study early human development or derive pluripotent hESC.     

In vivo confocal scanning laser microscopy: comparison of the reflectance and fluorescence mode by imaging human skin

Optical, noninvasive methods have become efficient in vivo tools in dermatological diagnosis and research. From these promising imaging techniques, only the confocal scanning laser microscopy (CSLM) provides visualization of subsurface skin structures with resolutions similar to those of light microscopy. Skin annexes, as well as cutaneous cells from different epidermal layers, can be distinguished excellently. Currently, two forms of application have been established in dermatological practice: the reflectance mode, predominantly in the clinical field, and the fluorescence mode in dermatological research. Differences in both methods exist in the preparative protocol, in maximum imaging depth and, particularly, in the gain of contrast extraction. The reflectance mode demonstrates naturally occurring tissue components, whereas the fluorescent CSLM achieves contrast by administering fluorescence dye, representing the dynamic distribution pattern of the dye's fluorescent emission. Therefore, the reflectance and fluorescent modes highlight various skin microstructures, providing dissimilar in vivo confocal images of the skin. This permits different predications and information on the state of the tissue. We report the advantages and disadvantages of both optical imaging modes. The comparison was drawn by scanning human skin in vivo. Representative images in varying depths were obtained and analyzed; preparation procedures are shown and discussed.     

Study on the lubrication mechanism of natural joints by confocal laser scanning microscopy

Natural joints have an excellent lubricating function, but the detailed mechanism is still unclear. To clarify this lubricating mechanism, we observed the behavior of the cartilage surface under the physiological loading condition with confocal laser scanning microscopy in normal and osteoarthritis (OA) cartilage rabbit specimens. Even with a considerable loading condition, in both natural and OA cartilage, the fluid pool area coexisted with the direct contact area. In the junction from the direct contact area to the fluid area, there was a third area with a liquid-crystal arrangement. In OA cartilage, these areas were generally irregular and small. These results suggest that a lubrication system in the fluid phase, such as squeeze film lubrication, might work under severe pressure in normal cartilage, and hyaluronic acid macromolecules in the synovial fluid might form a liquid-crystal structure and support pressure on the cartilage surface, whereas these systems did not affect the OA cartilage.     

Fast intracellular motion in the living cell by video rate reflection confocal laser scanning microscopy

Fast intracellular motion (FIM) was first revealed by back scattered light (BSL) imaging in video rate confocal scanning laser microscopy (VRCSLM), beyond the limits of spatial and temporal resolution obtainable with conventional optical microscopy. BSL imaging enabled visualisation of intra and extracellular motion with resolution in space down to 0.2 μm and in time to 1/25th of a second. Mapping the cell space at 0.2 μm×0.2 μm (XY = in instantaneous best focal plane)×0.5 μm (Z = height/depth, optic axis direction) volume steps revealed a communication layer above the known contact layer and an integrated dynamic spatial network (IDSN) towards the cell centre. FIM was originally observed as localised quasichaotic dancing (dithering) or reflecting patches/spots in the cell centre, faster in the darker nuclear space. Later, a second type of FIM was recognised which differed by the presence of a varied proportion of centrifugal and centripetal directional movements and/or jumping of patches/spots in the cell centre and outside the nuclear space. The first type is characteristic for cells in slightly adverse conditions while the second type has so far only been found in eutrophic cells. Temporal speeding up and coarsening of FIM, followed by slowing and eventually cessation at cell death, was found on exposure to strong stressors. It was concluded that the state of FIM provides instantaneous information about individual cell reactions to actual treatment and about cell survival. A putative switch between the first and second type FIM could be considered as an indicator of timing of cellular processes. The significance of FIM for the biology of the cell is seen in the rapid assessment of the condition of an individual live cell investigated by combination of various methods. Requirements for further development of this approach are outlined.     

Bound fibrinogen distribution on stimulated platelets. Examination by confocal scanning laser microscopy.

Previous studies have suggested that qualitative changes in platelet bound fibrinogen modulate platelet aggregation. The present study used confocal scanning laser microscopy to further evaluate post-ligand binding events over a 60-minute time course. When fluorescein isothiocyanate (FITC)-streptavidin was added to ADP-stimulated platelets 1 minute after biotinylated fibrinogen binding at 22 degrees C, bound fibrinogen was found in variously sized patches on the cell surface. When streptavidin was added 60 minutes later, bound fibrinogen had been cleared from the platelet surface and was observed in clusters penetrating into platelets to various extents. ADP-activated platelets did not stain with a monoclonal antibody against CD62 suggesting that platelets were not permeabilized during the experiment and had not released alpha-granules. Additional studies using either biotinylated fibrinogen that had been prelabeled with FITC-streptavidin or FITC-labeled fibrinogen revealed similar patterns of platelet-associated fibrinogen clearance and redistribution. Pretreatment of platelets with cytochalasin D prevented this redistribution. Dual labeling experiments using biotinylated fibrinogen and FITC-streptavidin as well as a monoclonal anti-GPIIIa antibody labeled with rhodamine-conjugated anti-mouse IgG demonstrated the co-localization of fibrinogen and GPIIIa. Similar observations were made with fibrinogen bound to thrombin-stimulated platelets. In contrast, fibronectin bound to thrombin-activated platelets retained a predominantly surface membrane distribution under identical experimental conditions. Since surface-cleared fibrinogen was accessible to exogenous FITC-streptavidin under conditions that did not lead to platelet permeabilization, the data suggest fibrinogen deposition in compartments that are accessible to the extracellular milieu. This is consistent with the ability of exogenous plasmin to completely remove cleared fibrinogen pools without detectable fibrinogen reexpression on the platelet surface or alpha-granule secretion. The data provide morphological evidence for the selective, GPIIb-IIIa mediated, actin-dependent clearance of bound fibrinogen from the activated platelet surface, suggesting a mechanism for preventing and limiting thrombus development.     

Abnormal cleavage and developmental arrest of human preimplantation embryos in vitro

Despite improvements in culture conditions and laboratory techniques still only about 50% of human embryos reach the blastocyst stage of development in vitro. While many factors influence embryo development, aberrant cleavage divisions have only recently been shown to directly affect the genome in individual cells of human embryos resulting in chromosome loss, mosaicism and cell arrest. In this article we review the current literature in the area of aberrant cleavage in human embryos and its effect on blastocyst development. Further to this, we propose a series of common abnormal cleavage events, with particular attention to timing and frequency, and illustrate how these might influence a number of different embryo fates.     

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.     

The chromosome constitution of human preimplantation embryos fertilized in vitro

The chromosome constitution of five haploid, 178 diploid and11 triploid embryos fertilized in vitro was determined afterfixation on day 2 or day 3 of development. Karyotype analysisof 178 diploid embryos revealed abnormalities in 40 (22.5%)cases: 34 (19.1%) aneuploids, four (2.2%) mosaic embryos andtwo (1.1%) structural anomalies were identified. The majorityof aneuploid karyotypes (28/34) involved a single chromosomebut six embryos had aneuploidy of two or three chromosomes.The E group was most frequently involved in aneuploid karyotypes(10/23 hyperdiploid embryos) and trisomy 16, the most commonsingle anomaly in diploid embryos, was detected in 2.2% (4/178)of cases. Only one case of sex chromosome monosomy was identified.An excess of female karyotypes was detected in abnormal cases(sex ratio 0.48); this ratio was significantly (p< 0.05)different from that observed in normal cases (74: 64, XY: XX).The incidence of aneuploidy increased with maternal age butthis did not reach statistical significance. Embryo morphologyand growth rate, assessed by embryo development rating (EDR),did not distinguish between normal (mean score 7.9; mean EDR96.1) and aneuploid (mean score 8.1; mean EDR, 92.1) embryos.Numbers of hyperploid (n = 17) and hypoploid (n= 11) embryos(non-mosaic cases involving single chromosomes) were not statisticallydifferent. The relative proportions of chromosomes involvedin trisomic karyotypes showed a remarkable similarity to thepattern in spontaneous abortions. Pronuclear status was an unreliablepredictor of ploidy. Small numbers of karyotyped triploid embryosrevealed equal proportions of XXX, XXY and XYY embryos     

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。     

Identification and characterization of rat orbicularis oculi motoneurons using confocal laser scanning microscopy

 The eyeblink reflex is one of the most extensively studied behaviors in mammals. The active downward force that causes lid closure is controlled by the orbicularis oculi (OO) muscle. To augment our studies on the neurophysiology and plasticity of the rat eyeblink circuit, here we present the first anatomical paper to focus exclusively on identifying and characterizing the OO motoneurons of the rat facial motor nucleus (FMN). One thousand and twenty-nine cells from four animals were retrogradely labeled by injecting the OO muscle with HRP and were imaged conventionally. One hundred and one cells from five animals were labeled by injecting the OO muscle with a 3000 mol. wt. fluorescent dextran and were imaged using confocal laser scanning microscopy (CLSM). The latter method resulted in little tissue shrinkage, bright labeling, and excellent resolution of the soma, dendrites, and axon. Furthermore, it is a histologically simple alternative to HRP for retrograde labeling from the neuromuscular junction. Both methods revealed that the OO motoneurons were distributed over the entire length of the FMN, that they were concentrated along the dorsal crest of the nucleus, and that they were less numerous in the extreme rostral and caudal regions. As measured using the CLSM method, cell body areas were highly variable, ranging from 317 to 1500 μm², but there was no size gradient along the rostrocaudal extent of the FMN. The neurons exhibited seven primary dendrites on average, which gave rise to bifurcating and even trifurcating secondary dendrites. Using the HRP method, the estimated area of OO motoneurons ranged from 161 to 1381 μm². The combined methods furnished a detailed characterization of the number, spatial distribution, and morphology of rat OO motoneurons. Moreover, these methods provide a useful way to analyze the circuitry that modulates the rat eyeblink. Received: 18 September 1996 / Accepted: 10 March 1997     

Immunohistochemical detection of three serum protease inhibitors in mouse skeletal muscle by confocal laser scanning microscopy

The tissue-associated counterpart of some plasmatic protease inhibitors has been studied in mouse skeletal muscle by combining immunoperoxidase confocal microscopy and Western blot analysis. To remove serum contamination all experiments were performed on C57 BL/10 adult mice perfused extensively with physiological solution under deep anesthesia. The following serum inhibitors were investigated in skeletal muscle by immunoperoxidase staining: alpha-2-macroglobulin (alpha2M), antithrombin III (ATIII) and inter-alpha-trypsin inhibitor (ITI). The resulting localization patterns were analysed by laser transmittance scanning at 488 nm using a confocal microscope. Images obtained from a series of optical sections were then digitally intensified by a computerized program, allowing detection of even negligible amounts of immunoreaction product. In all muscles examined (soleus and extensor digitorum longus mm.) an extracellular (endomysial) localization was apparent for all inhibitors. By contrast remarkable differences were observed for the intracellular component: in fact alpha2M was present in about a half of the muscle fibers; ATIII was present inside all fibers; intracellular ITI was completely absent. Western blotting analysis of muscle homogenate was performed to biochemically characterize the above immunoreactivities. In preliminary experiments alpha2M-related immunoreactivity could not be found in the soluble fraction of perfused muscle, confirming an absence of serum contamination after in vivo perfusion. By contrast experiments on detergent-solubilized extracts (0.3% Triton X-100) revealed that tissue-bound alpha2M consisted of two main bands (168-166 KDa) and a minor component (35 KDa); ATIII of a single band (50 KDA); ITI of four bands (180, 50, 45, 40 KDa). These results confirmed that the specific immunoreactivities visualized by morphological techniques corresponded to muscle-associated plasmatic inhibitors. The present data suggest that in mouse skeletal muscle i) numerous tissue-associated plasmatic inhibitors may protect the extracellular matrix from an excess of proteolysis; ii) a more restricted set of inhibitors may be also involved in the down-regulation of intracellular proteolytic processes.     

Analysis of tissue and subcellular localization of mammalian diamine oxidase by confocal laser scanning fluorescence microscopy

Inflammation Research -     

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.