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Histochemical assessment on the cellular interplay of vascular endothelial cells and septoclasts during endochondral ossification in mice 组织化学评价小鼠软骨内骨化过程中血管内皮细胞和破隔膜细胞的相互作用
IF 1.8 4区 工程技术 Pub Date : 2020-11-01 DOI: 10.1093/jmicro/dfaa047
Erika Tsuchiya;Tomoka Hasegawa;Hiromi Hongo;Tomomaya Yamamoto;Miki Abe;Taiji Yoshida;Shen Zhao;Kanako Tsuboi;Nobuyuki Udagawa;Paulo Henrique Luiz de Freitas;Minqi Li;Yoshimasa Kitagawa;Norio Amizuka
This study was aimed to verify the cellular interplay between vascular endothelial cells and surrounding cells in the chondro-osseous junction of murine tibiae. Many CD31-positive endothelial cells accompanied with Dolichos Biflorus Agglutinin lectin-positive septoclasts invaded into the hypertrophic zone of the tibial epiphyseal cartilage. MMP9 immunoreactive cytoplasmic processes of vascular endothelial cells extended into the transverse partitions of cartilage columns. In contrast, septoclasts included several large lysosomes which indicate the incorporation of extracellular matrices despite no immunopositivity for F4/80—a hallmark of macrophage/monocyte lineage. In addition, septoclasts were observed in c-fos-/- mice but not in Rankl-/- mice. Unlike c-fos-/- mice, Rankl-/- mice showed markedly expanded hypertrophic zone and the irregular shape of the chondro-osseous junction. Immunoreactivity of platelet-derived growth factor-bb, which involved in angiogenic roles in the bone, was detected in not only osteoclasts but also septoclasts at the chondro-osseous junction. Therefore, septoclasts appear to assist the synchronous vascular invasion of endothelial cells at the chondro-osseous junction. Vascular endothelial cells adjacent to the chondro-osseous junction possess endomucin but not EphB4, whereas those slightly distant from the chondro-osseous junction were intensely positive for both endomucin and EphB4, while being accompanied with ephrinB2-positive osteoblasts. Taken together, it is likely that vascular endothelial cells adjacent to the chondro-osseous junction would interplay with septoclasts for synchronous invasion into the epiphyseal cartilage, while those slightly distant from the chondro-osseous junction would cooperate with osteoblastic activities presumably by mediating EphB4/ephrinB2. Mini-abstract Our original article demonstrated that vascular endothelial cells adjacent to the chondro-osseous junction would interplay with septoclasts for synchronous invasion into the epiphyseal cartilage, while those slightly distant from the chondro-osseous junction would cooperate with osteoblastic activities presumably by mediating EphB4/ephrinB2. (A figure that best represents your paper is Fig. 5c)
本研究旨在验证小鼠胫骨软骨-骨连接处血管内皮细胞和周围细胞之间的细胞相互作用。许多CD31阳性的内皮细胞伴随着Dolichos Biflorus凝集素阳性的隔膜破坏者侵入胫骨骨骺软骨的肥大区。MMP9免疫反应阳性的血管内皮细胞胞质突起延伸到软骨柱的横向分区。相反,尽管F4/80没有免疫阳性,但破隔膜剂包括几个大的溶酶体,这表明细胞外基质的结合——这是巨噬细胞/单核细胞谱系的标志。此外,在c-fos小鼠中观察到了隔膜破坏剂,但在Rankl-/-小鼠中没有观察到。与c-fos小鼠不同,Rankl-/-小鼠的肥大区明显扩大,软骨-骨连接处形状不规则。参与骨血管生成作用的血小板衍生生长因子bb的免疫反应性不仅在破骨细胞中检测到,而且在软骨-骨连接处的破隔膜细胞中也检测到。因此,破隔膜剂似乎有助于软骨-骨交界处内皮细胞的同步血管侵袭。软骨-骨交界处附近的血管内皮细胞具有内粘蛋白,但不具有EphB4,而那些稍微远离软骨-骨接合处的细胞对内粘蛋白和EphB4均呈强阳性,同时伴有ephrinB2阳性的成骨细胞。总之,软骨-骨交界处附近的血管内皮细胞可能会与破隔膜细胞相互作用,同步侵入骨骺软骨,而那些稍微远离软骨-骨接合处的细胞可能会通过介导EphB4/ephrinB2与成骨细胞活性协同作用。迷你摘要我们的原始文章证明,软骨-骨交界处附近的血管内皮细胞将与破隔膜细胞相互作用,同步侵入骨骺软骨,而那些离软骨-骨接合处稍远的细胞可能通过介导EphB4/ephrinB2与成骨细胞活动协同作用。(最能代表你论文的数字是图5c)
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引用次数: 9
Three-dimensional culture of a pancreatic cancer cell line, SUIT-58, with air exposure can reflect the intrinsic features of the original tumor through electron microscopy 暴露在空气中的胰腺癌症细胞系SUITE-58的三维培养可以通过电子显微镜反映原始肿瘤的内在特征
IF 1.8 4区 工程技术 Pub Date : 2020-11-01 DOI: 10.1093/jmicro/dfaa046
Nobuyasu Takahashi;Fumiyo Aoyama;Akira Sawaguchi
Mini-abstract: Application of a three-dimensional culture system with air exposure facilitates the formation of large cell spheres possessing cribriform glands and producing mucin in the collagen gel. Transmission electron microscopy revealed the formation of microvilli and junctional complexes at the apical side of the cell. This study aimed to reproduce the characteristics of original adenocarcinoma tumors in vitro. The pancreatic cell line, SUIT-58, derived from a moderately differentiated adenocarcinoma of metastatic pancreatic cancer was used. The cells have a sheet structure in conventional cell culture without forming glands or exhibiting mucin production in the lumen. First, the necessity of scaffolds to create an adenocarcinoma-like microenvironment for SUIT-58 pancreatic cancer cells was assessed. Compared with conventional culture plates, the use of type I collagen as a scaffold played an important role in the formation of densely congested microvilli, as observed through scanning electron microscopy. As gland formation is one of the features of adenocarcinoma, we also assessed gland formation. Use of a recently developed three-dimensional culture system with air exposure resulted in the formation of large cell spheres possessing cribriform glands, which released mucin into the lumen. Transmission electron microscopy also revealed the formation of microvilli in the lumen of the glands and junctional complex at the intercellular part, which were similar to those observed in xenografts. These findings indicate that an in vitro three-dimensional culture system with air exposure reflects the intrinsic features of the original tumor, suggesting that this culture system could be useful for preliminary research of certain cancers.
摘要:应用暴露在空气中的三维培养系统有助于形成具有筛状腺的大细胞球,并在胶原凝胶中产生粘蛋白。透射电子显微镜显示细胞顶端形成微绒毛和连接复合体。本研究旨在体外再现原发性腺癌肿瘤的特征。使用来源于转移性癌症的中分化腺癌的胰腺细胞系SUI-58。在常规细胞培养中,细胞具有片状结构,而在管腔中不形成腺体或表现出粘蛋白产生。首先,评估了支架为SUITE-58胰腺癌症细胞创建腺癌样微环境的必要性。通过扫描电子显微镜观察到,与传统培养板相比,使用I型胶原作为支架在密集充血微绒毛的形成中发挥了重要作用。由于腺体形成是腺癌的特征之一,我们也评估了腺体的形成。使用最近开发的暴露在空气中的三维培养系统可以形成具有筛状腺的大细胞球,从而将粘蛋白释放到管腔中。透射电子显微镜还显示,腺体内腔中形成了微绒毛,细胞间部分形成了连接复合体,这与异种移植物中观察到的类似。这些发现表明,暴露在空气中的体外三维培养系统反映了原始肿瘤的内在特征,表明该培养系统可用于某些癌症的初步研究。
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引用次数: 1
Phase detection limits in off-axis electron holography from pixelated detectors: gain variations, geometric distortion and failure of reference-hologram correction 像素化探测器离轴电子全息术的相位检测极限:增益变化、几何畸变和参考全息图校正失败
IF 1.8 4区 工程技术 Pub Date : 2020-11-01 DOI: 10.1093/jmicro/dfaa044
Martin Hÿtch;Christophe Gatel
We investigate the effect that recording off-axis electron holograms on pixelated detectors, such as charge-coupled devices (CCD) and direct-detection devices (DDD), can have on measured amplitudes and phases. Theory will be developed for the case of perfectly uniform interference fringes illuminating an imperfect detector with gain variations and pixel displacements. We will show that both these types of defect produce a systematic noise in the phase images that depends on the position of the holographic fringes with respect to the detector. Subtracting a reference hologram from the object hologram will therefore not remove the phase noise if the initial phases of the two holograms do not coincide exactly. Another finding is that pi-shifted holograms are much less affected by gain variations but show no improvement concerning geometric distortions. The resulting phase errors will be estimated and simulations presented that confirm the theoretical developments.
我们研究了在像素化探测器(如电荷耦合器件(CCD)和直接探测器件(DDD))上记录离轴电子全息图对测量振幅和相位的影响。将针对具有增益变化和像素位移的完美均匀干涉条纹照射不完美检测器的情况来发展理论。我们将证明,这两种类型的缺陷在相位图像中都会产生系统噪声,该噪声取决于全息条纹相对于检测器的位置。因此,如果两个全息图的初始相位不完全一致,那么从物体全息图中减去参考全息图将不会去除相位噪声。另一个发现是π移位全息图受增益变化的影响要小得多,但在几何失真方面没有改善。将对由此产生的相位误差进行估计,并进行模拟,以证实理论发展。
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引用次数: 1
Three-dimensional structure analysis of melanocytes and keratinocytes in senile lentigo 老年性扁豆黑色素细胞和角质形成细胞的三维结构分析
IF 1.8 4区 工程技术 Pub Date : 2020-11-01 DOI: 10.1093/jmicro/dfaa054
Yuki Mizutani;Mika Yamashita;Rie Hashimoto;Toru Atsugi;Akemi Ryu;Akinobu Hayashi;Yukiko Rikimaru-Nishi;Keisuke Ohta
Senile lentigo or age spots are hyperpigmented macules of skin that commonly develop following long-term exposure to ultraviolet radiation. This condition is caused by accumulation of large numbers of melanosomes (melanin granules) produced by melanocytes within neighboring keratinocytes. However, there is still no consensus regarding the melanosome transfer mechanism in senile lentigo. To date, most pathohistological studies of skin have been two-dimensional and do not provide detailed data on the complex interactions of the melanocyte–keratinocyte network involved in melanosome transfer. We performed a three-dimensional reconstruction of the epidermal microstructure in senile lentigo using three different microscopic modalities to visualize the topological melanocyte–keratinocyte relationship and melanosome distribution. Confocal laser microscopy images showed that melanocyte dendritic processes are more frequently branched and elongated in senile lentigo skin than in normal skin. Serial transmission electron micrographs showed that dendritic processes extend into intercellular spaces between keratinocytes. Focused ion beam-scanning electron micrographs showed that dendritic processes in senile lentigo encircle adjacent keratinocytes and accumulate large numbers of melanosomes. Moreover, melanosomes transferred to keratinocytes are present not only in the supranuclear area but throughout the perinuclear area except on the basal side. The use of these different microscopic methods helped to elucidate the three-dimensional morphology and topology of melanocytes and keratinocytes in senile lentigo. We show that the localization of melanosomes in dendritic processes to the region encircling recipient keratinocytes contributes to efficient melanosome transfer in senile lentigo.
老年性雀斑或老年斑是皮肤上的色素沉着斑,通常在长期暴露于紫外线辐射后形成。这种情况是由邻近角质形成细胞内黑色素细胞产生的大量黑色素体(黑色素颗粒)积聚引起的。然而,对于老年性扁豆的黑色素体转移机制,目前还没有达成共识。到目前为止,大多数皮肤的病理学研究都是二维的,并且没有提供关于参与黑色素体转移的黑色素细胞-角质形成细胞网络复杂相互作用的详细数据。我们使用三种不同的显微镜模式对老年性扁豆的表皮微观结构进行了三维重建,以观察拓扑黑素细胞-角质形成细胞的关系和黑素体的分布。共焦激光显微镜图像显示,与正常皮肤相比,老年性雀斑皮肤中的黑素细胞树突突起更频繁地分支和延长。连续的透射电子显微照片显示树突突起延伸到角质形成细胞之间的细胞间隙。聚焦离子束扫描电子显微照片显示,老年扁豆的树突突起包围相邻的角质形成细胞并积累大量黑素体。此外,转移到角质形成细胞的黑素体不仅存在于核上区域,而且存在于除基底侧外的整个核周区域。使用这些不同的显微镜方法有助于阐明老年性扁豆中黑色素细胞和角质形成细胞的三维形态和拓扑结构。我们发现,黑素体在树突过程中定位于受体角质形成细胞周围的区域,有助于老年性扁豆中黑素体的有效转移。
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引用次数: 6
Wave field reconstruction and phase imaging by electron diffractive imaging 用电子衍射成像实现波场重建和相位成像
IF 1.8 4区 工程技术 Pub Date : 2020-11-01 DOI: 10.1093/jmicro/dfaa063
Jun Yamasaki
In electron diffractive imaging, the phase image of a sample is reconstructed from its diffraction intensity through iterative calculations. The principle of this method is based on the Fourier transform relation between the real-space wave field transmitted by the sample and its Fraunhofer diffraction wave field. Since Gerchberg’s experimental work in 1972, various advancements have been achieved, which have substantially improved the quality of the reconstructed phase images and extended the applicable range of the method. In this review article, the principle of diffractive imaging, various experimental processes using electron beams and application to specific samples are explained in detail.
在电子衍射成像中,样品的相位图像是通过迭代计算从其衍射强度重建的。该方法的原理是基于样品传输的真实空间波场与其Fraunhofer衍射波场之间的傅立叶变换关系。自1972年Gerchberg的实验工作以来,已经取得了各种进展,大大提高了重建相位图像的质量,并扩大了该方法的适用范围。在这篇综述文章中,详细解释了衍射成像的原理、使用电子束的各种实验过程以及在特定样品中的应用。
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引用次数: 0
Interference and interferometry in electron holography 电子全息术中的干涉与干涉
IF 1.8 4区 工程技术 Pub Date : 2020-11-01 DOI: 10.1093/jmicro/dfaa033
Ken Harada
This paper reviews the basics of electron holography as an introduction of the holography part of this special issue in Microscopy. We discuss the general principle of holography and interferometry regarding measurements and analyses of phase distributions, first using the optical holography. Next, we discuss physical phenomena peculiar to electron waves that cannot be realized by light waves and principles of electromagnetic field detection and observation methods. Furthermore, we discuss the interference optical systems of the electron waves and their features, and methods of reconstruction of the phase information from electron holograms, which are essential for realization of practical electron holography. We note that following this review application of electron holography will be discussed in detail in the papers of this special issue.
本文综述了电子全息术的基本原理,并介绍了本期《显微镜》特刊的全息术部分。我们首先利用光学全息术讨论了全息术和干涉测量的一般原理,包括相位分布的测量和分析。接下来,我们将讨论光波无法实现的电子波特有的物理现象以及电磁场检测和观测方法的原理。此外,我们还讨论了电子波的干涉光学系统及其特征,以及从电子全息图中重建相位信息的方法,这对实现实用的电子全息至关重要。我们注意到,在这篇综述之后,电子全息术的应用将在本期特刊的论文中详细讨论。
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引用次数: 9
Evaluation of accuracy in the determination of crystal structure factors using large-angle convergent-beam electron diffraction patterns 用大角度会聚光束电子衍射图测定晶体结构因子的准确性评价
IF 1.8 4区 工程技术 Pub Date : 2020-11-01 DOI: 10.1093/jmicro/dfaa041
Daisuke Morikawa;Kenji Tsuda
The accuracy of electron density distribution analysis using large-angle convergent-beam electron diffraction (LACBED) patterns is evaluated for different convergence angles. An orbital ordered state of FeCr2O4 is used as an example of the analysis. Ideal orbital-ordered and non-ordered states are simulated by using orbital scattering factors. LACBED patterns calculated for the orbital-ordered state were used as hypothetical experimental data sets. Electron density distribution of the Fe 3d orbitals has been successfully reconstructed with a higher accuracy from LACBED patterns with convergence angles larger than 15.2 mrad, which is 4 times as large as that for conventional convergent-beam electron diffraction patterns. Excitation of particular Bloch waves with the aid of LACBED patterns has a key role in the accurate analysis of electron density distributions.
评估了在不同会聚角下使用大角度会聚光束电子衍射(LACBED)图进行电子密度分布分析的准确性。使用FeCr2O4的轨道有序态作为分析的例子。利用轨道散射因子模拟了理想轨道有序态和非有序态。为轨道有序状态计算的LACBED模式被用作假设的实验数据集。从会聚角大于15.2mrad的LACBED图中成功地以更高的精度重建了Fe3d轨道的电子密度分布,这是传统会聚束电子衍射图的4倍。在LACBED模式的帮助下激发特定的布洛赫波在精确分析电子密度分布中起着关键作用。
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引用次数: 2
Sandwich freezing device for rapid freezing of viruses, bacteria, yeast, cultured cells and animal and human tissues in electron microscopy 用于电子显微镜下快速冷冻病毒、细菌、酵母、培养细胞、动物和人体组织的三明治冷冻装置
IF 1.8 4区 工程技术 Pub Date : 2020-11-01 DOI: 10.1093/jmicro/dfaa049
Masashi Yamaguchi;Masaki Taguchi;Katsuyuki Uematsu;Azusa Takahashi-Nakaguchi;Michiyo Sato-Okamoto;Hiroji Chibana
We have been using sandwich freezing of living yeast and bacteria followed by freeze-substitution for observing close-to-native ultrastructure of cells. Recently, sandwich freezing of glutaraldehyde-fixed cultured cells and human tissues have been found to give excellent preservation of ultrastructure of cells and tissues. These studies, however, have been conducted using a handmade sandwich freezing device and have been limited in a few laboratories. To spread the use of this method to other laboratories, we fabricated and commercialized a new sandwich freezing device. The new device is inexpensive, portable and sterilizable. It can be used to rapid-freeze viruses, bacteria, yeast, cultured cells and animal and human tissues to a depth of 0.2 mm if tissues are prefixed with glutaraldehyde. The commercial availability of this device will expand application of rapid freezing to wide range of biological materials.
我们一直在使用活酵母和细菌的三明治冷冻,然后用冷冻替代来观察接近天然细胞的超微结构。最近,戊二醛固定培养的细胞和人体组织的三明治冷冻被发现可以极好地保存细胞和组织的超微结构。然而,这些研究是使用手工制作的三明治冷冻装置进行的,在少数实验室受到限制。为了将这种方法推广到其他实验室,我们制造并商业化了一种新的三明治冷冻装置。这种新设备价格低廉,便于携带和消毒。如果组织前面有戊二醛,它可以用于将病毒、细菌、酵母、培养细胞、动物和人类组织快速冷冻至0.2毫米的深度。该装置的商业可用性将扩大快速冷冻在广泛生物材料中的应用。
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引用次数: 3
Introduction to ‘electron interference microscopy’ “电子干涉显微镜”简介
IF 1.8 4区 工程技术 Pub Date : 2020-11-01 DOI: 10.1093/jmicro/dfaa067
Ken Harada;Tadahiro Kawasaki
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引用次数: 0
Holography: application to high-resolution imaging 全息:在高分辨率成像中的应用
IF 1.8 4区 工程技术 Pub Date : 2020-11-01 DOI: 10.1093/jmicro/dfaa050
Takeshi Kawasaki;Yoshio Takahashi;Toshiaki Tanigaki
Electron holography was invented for correcting aberrations of the lenses of electron microscopes. It was used to observe the atomic arrangements in crystals after decades of research. Then it was combined with a hardware aberration corrector to enable high-resolution and high-precision analysis. Its applications were further extended to magnetic observations with sub-nanometer resolution. High-resolution electron holography has become a powerful technique for observing electromagnetic distributions in functional materials.
电子全息术是为了校正电子显微镜透镜的像差而发明的。经过几十年的研究,它被用来观察晶体中的原子排列。然后将其与硬件像差校正器相结合,实现高分辨率和高精度的分析。它的应用进一步扩展到亚纳米分辨率的磁性观测。高分辨率电子全息术已成为观测功能材料中电磁分布的一种强大技术。
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引用次数: 1
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