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TEM and STEM-EDS evaluation of metal nanoparticle encapsulation in GroEL/GroES complexes according to the reaction mechanism of chaperonin 根据伴侣蛋白的反应机理对GroEL/GroES复合物中金属纳米粒子包封的TEM和STEM-EDS评估
IF 1.8 4区 工程技术 Pub Date : 2021-06-01 DOI: 10.1093/jmicro/dfaa064
Hiromi Yoda;Ayumi Koike-Takeshita
Escherichia coli chaperonin GroEL, which is a large cylindrical protein complex comprising two heptameric rings with cavities of 4.5 nm each in the center, assists in intracellular protein folding with the aid of GroES and adenosine triphosphate (ATP). Here, we investigated the possibility that GroEL can also encapsulate metal nanoparticles (NPs) up to ∼5 nm in diameter into the cavities with the aid of GroES and ATP. The slow ATP-hydrolyzing GroELD52A/D398A mutant, which forms extremely stable complexes with GroES (half-time of ∼6 days), made it possible to analyze GroEL/GroES complexes containing metal NPs. Scanning transmission electron microscopy–energy-dispersive X-ray spectroscopy analysis proved distinctly that FePt NPs and Au NPs were encapsulated in the GroEL/GroES complexes. Dynamic light scattering measurements showed that the NPs in the GroEL/GroES complex were able to maintain their dispersibility in solution. We previously described that the incubation of GroEL and GroES in the presence of ATP·BeFx and adenosine diphosphate·BeFx resulted in the formation of symmetric football-shaped and asymmetric bullet-shaped complexes, respectively. Based on this knowledge, we successfully constructed the football-shaped complex in which two compartments were occupied by Pt or Au NPs (first compartment) and FePt NPs (second compartment). This study showed that metal NPs were sequentially encapsulated according to the GroEL reaction in a step-by-step manner. In light of these results, chaperonin can be used as a tool for handling nanomaterials.
大肠杆菌伴侣蛋白GroEL是一种大的圆柱形蛋白质复合物,包括两个中心各有4.5nm空腔的七聚环,在GroES和三磷酸腺苷(ATP)的帮助下帮助细胞内蛋白质折叠。在这里,我们研究了GroEL也可以在GroES和ATP的帮助下将直径高达~5 nm的金属纳米颗粒(NP)封装到空腔中的可能性。缓慢ATP水解的GroELD52A/D398A突变体与GroES形成极其稳定的复合物(半衰期约6天),使分析含有金属NP的GroEL/GroES复合物成为可能。扫描透射电子显微镜-能量色散X射线光谱分析清楚地证明,FePt NPs和Au NPs被包裹在GroEL/GroES复合物中。动态光散射测量表明,GroEL/GroES复合物中的NP能够保持其在溶液中的分散性。我们之前描述过,GroEL和GroES在ATP·BeFx和二磷酸腺苷·BeFx存在下的孵育分别导致对称足球形和不对称子弹形复合物的形成。基于这些知识,我们成功地构建了足球形状的复合体,其中两个隔间被Pt或Au NP(第一隔间)和FePt NP(第二隔间)占据。该研究表明,金属NP是根据GroEL反应以逐步的方式顺序封装的。鉴于这些结果,伴侣蛋白可以用作处理纳米材料的工具。
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引用次数: 1
Phase imaging dislocations using diffracted beam interferometry 用衍射束干涉法对位错进行相位成像
IF 1.8 4区 工程技术 Pub Date : 2021-06-01 DOI: 10.1093/jmicro/dfaa066
Rodney Herring
A phase imaging method that measures the phase shift existing at a dislocation's core is described. The method uses the interference of two symmetrically diffracted beams on the optic axis by means of an electron biprism. Each diffracted beam carries half the phase of the dislocation core. When combined, the entire phase shift of the dislocation core is obtained.
描述了一种测量位错核处存在的相移的相位成像方法。该方法利用两个对称衍射光束在光轴上的电子双棱镜干涉。每个衍射光束携带位错核心的一半相位。当组合时,可以获得位错核心的整个相移。
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引用次数: 3
TEM observation of compacted DNA of Synechococcus elongatus PCC 7942 using DRAQ5 labeling with DAB photooxidation and osmium black DAB光氧化和锇黑DRAQ5标记细长聚球藻PCC 7942 DNA的透射电镜观察
IF 1.8 4区 工程技术 Pub Date : 2021-06-01 DOI: 10.1093/jmicro/dfaa058
Ilika Ghosh;Kimie Atsuzawa;Aoi Arai;Ryuzo Ohmukai;Yasuko Kaneko
To visualize the fine structure of compacted DNA of Synechococcus elongatus PCC 7942, which appears at a specific time in the regular light/dark cycle prior to cell division, ChromEM with some modifications was applied. After staining DNA with DRAQ5, the cells were fixed and irradiated by red laser in the presence of 3,3ʹ-diaminobenzidine and subsequently fixed with OsO4. A system with He–Ne laser (633 nm) was set up for efficient irradiation of the bacterial cells in aqueous solution. The compacted DNA was visualized by transmission electron microscopy, in ultrathin sections as electron dense staining by osmium black.
为了观察在细胞分裂前的规则光/暗周期中的特定时间出现的细长聚球藻PCC 7942的压实DNA的精细结构,应用了具有一些修饰的ChromEM。用DRAQ5染色DNA后,将细胞固定,并在3,3-二氨基联苯胺存在下用红色激光照射,随后用OsO4固定。建立了一个用He–Ne激光(633nm)有效照射水溶液中细菌细胞的系统。通过透射电子显微镜观察压实的DNA,在超薄切片中通过锇黑进行电子致密染色。
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引用次数: 1
Ultrastructural evidence for an unusual mode of ciliogenesis in mouse multiciliated epithelia 小鼠多纤毛上皮纤毛发生异常模式的超微结构证据
IF 1.8 4区 工程技术 Pub Date : 2021-06-01 DOI: 10.1093/jmicro/dfaa074
Keishi Narita;Sen Takeda
Multiciliogenesis is a cascading process for generating hundreds of motile cilia in single cells. In vertebrates, this process has been investigated in the ependyma of brain ventricles and the ciliated epithelia of the airway and oviduct. Although the early steps to amplify centrioles have been characterized in molecular detail, subsequent steps to establish multicilia have been relatively overlooked. Here, we focused on unusual cilia-related structures previously observed in wild-type mouse ependyma using transmission electron microscopy and analyzed their ultrastructural features and the frequency of their occurrence. In the ependyma, $sim$5% of cilia existed as bundles; while the majority of the bundles were paired, bundles of more than three cilia were also found. Furthermore, apical protrusions harboring multiple sets of axonemes were occasionally observed (0–2 per section), suggesting an unusual mode of ciliogenesis. In trachea and oviduct epithelia, ciliary bundles were absent, but protrusions containing multiple axonemes were observed. At the base of such protrusions, certain axonemes were completely enwrapped by membranes, whereas others remained incompletely enwrapped. These data suggested that the late steps of multiciliogenesis might include a unique process underlying the development of cilia, which is distinct from the ciliogenesis of primary cilia.
多纤毛发生是在单个细胞中产生数百个活动纤毛的级联过程。在脊椎动物中,这种过程已经在脑室的室管膜以及气道和输卵管的纤毛上皮中进行了研究。尽管扩增中心粒的早期步骤已经在分子细节上得到了表征,但随后建立多纤毛的步骤相对被忽视了。在这里,我们重点研究了先前在野生型小鼠室管膜中使用透射电子显微镜观察到的异常纤毛相关结构,并分析了它们的超微结构特征和发生频率。在室管膜中,$ sim$5%的纤毛以束状存在;虽然大多数纤毛束是成对的,但也发现了三个以上纤毛束。此外,偶尔观察到含有多组轴突的顶端突起(每个切片0-2个),这表明纤毛形成的模式不同寻常。在气管和输卵管上皮中,纤毛束不存在,但观察到含有多个轴突的突起。在这种突起的底部,某些轴突完全被膜包裹,而另一些则保持不完全包裹。这些数据表明,多细胞纤毛发生的后期可能包括纤毛发育的独特过程,这与初级纤毛的纤毛发生不同。
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引用次数: 2
Evaluation of depth of dislocation visibility in SEM electron channeling contrast imaging in Ti-6Al-4V alloy using serial sectioning method 用连续切片法评价Ti-6Al-4V合金SEM电子沟道对比成像中位错可见度的深度
IF 1.8 4区 工程技术 Pub Date : 2021-06-01 DOI: 10.1093/jmicro/dfaa060
Shigeto Yamasaki;Misaki Deguchi;Masatoshi Mitsuhara;Hideharu Nakashima;Yutaro Ota;Keiji Kubushiro
In this study, we conducted a quantitative evaluation of dislocation density by scanning electron microscopy electron channeling contrast imaging for α grains of a Ti-6Al-4V alloy deformed at room temperature. The depth of visibility of dislocations is experimentally measured as 140 to 160 nm by a serial sectioning observation. This result is compared with the theoretical value and applied to evaluate dislocation density. These factors confirm that the theoretically calculated value of the depth of visibility, at 5 to 6 times the extinction distance, is valid for the hexagonal close-packed Ti alloy.
在本研究中,我们通过扫描电子显微镜电子沟道对比成像对室温下变形的Ti-6Al-4V合金的α晶粒的位错密度进行了定量评估。位错的可见深度通过连续切片观察实验测量为140至160nm。将该结果与理论值进行了比较,并应用于位错密度的评价。这些因素证实,在消光距离的5到6倍处,可视深度的理论计算值对于六方紧密堆积的Ti合金是有效的。
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引用次数: 0
Photobleaching reduction in modulated super-resolution microscopy 调制超分辨率显微镜中的光漂白减少
IF 1.8 4区 工程技术 Pub Date : 2021-06-01 DOI: 10.1093/jmicro/dfaa062
Jafar H Ghithan;Jennifer M Noel;Thomas J Roussel;Maureen A McCall;Bruce W Alphenaar;Sergio B Mendes
Important breakthroughs in far-field imaging techniques have been made since the first demonstrations of stimulated emission depletion (STED) microscopy. To date, the most straightforward and widespread deployment of STED microscopy has used continuous wave (CW) laser beams for both the excitation and depletion of fluorescence emission. A major drawback of the CW STED imaging technique has been photobleaching effects due to the high optical power needed in the depletion beam to reach sub-diffraction resolution. To overcome this hurdle, we have applied a synchronous detection approach based on modulating the excitation laser beam, while keeping the depletion beam at CW operation, and frequency filtering the collected signal with a lock-in amplifier to record solely the super-resolved fluorescence emission. We demonstrate here that such approach allows an important reduction in the optical power of both laser beams that leads to measurable decreases in photobleaching effects in STED microscopy. We report super-resolution images with relatively low powers for both the excitation and depletion beams. In addition, typical unwanted scattering effects and background signal generated from the depletion beam, which invariably arises from mismatches in refractive index in the material composing the sample, are largely reduced by using the modulated STED approach. The capability of acquiring super-resolution images with relatively low power is quite relevant for studying a variety of samples, but particularly important for biological species as exemplified in this work.
自首次演示受激发射耗尽(STED)显微镜以来,远场成像技术取得了重要突破。迄今为止,STED显微镜最直接、最广泛的部署已经使用连续波(CW)激光束来激发和耗尽荧光发射。CW STED成像技术的一个主要缺点是由于耗尽光束中达到亚衍射分辨率所需的高光功率而产生的光漂白效应。为了克服这一障碍,我们应用了一种同步检测方法,该方法基于调制激发激光束,同时将耗尽光束保持在CW操作,并用锁定放大器对收集的信号进行频率滤波,以仅记录超分辨荧光发射。我们在这里证明,这种方法可以显著降低两个激光束的光功率,从而导致STED显微镜中的光漂白效应显著降低。我们报道了激发光束和耗尽光束都具有相对低功率的超分辨率图像。此外,通过使用调制STED方法,从耗尽光束产生的典型的不想要的散射效应和背景信号(其总是由组成样品的材料中的折射率失配引起)被大大减少。以相对较低的功率获取超分辨率图像的能力与研究各种样本非常相关,但对本工作中举例说明的生物物种尤其重要。
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引用次数: 1
Denoising of series electron holograms using tensor decomposition 用张量分解对串联电子全息图进行去噪
IF 1.8 4区 工程技术 Pub Date : 2021-06-01 DOI: 10.1093/jmicro/dfaa057
Yuki Nomura;Kazuo Yamamoto;Satoshi Anada;Tsukasa Hirayama;Emiko Igaki;Koh Saitoh
In this study, a noise-reduction technique for series low-dose electron holograms using tensor decomposition is demonstrated through simulation. We treated an entire dataset of the series holograms with Poisson noise as a third-order tensor, which is a stack of 2D holograms. The third-order tensor, which is decomposed into a core tensor and three factor matrices, is approximated as a lower-rank tensor using only noise-free principal components. This technique is applied to simulated holograms by assuming a p-n junction in a semiconductor sample. The peak signal-to-noise ratios of the holograms and the reconstructed phase maps have been improved significantly using tensor decomposition. Moreover, the proposed method was applied to a more practical situation of time-resolved in situ electron holography by considering a nonuniform fringe contrast and fringe drift relative to the sample. The accuracy and precision of the reconstructed phase maps were quantitatively evaluated to demonstrate its effectiveness for in situ experiments and low-dose experiments on beam-sensitive materials.
在本研究中,通过模拟演示了一种使用张量分解的串联低剂量电子全息图降噪技术。我们将具有泊松噪声的系列全息图的整个数据集处理为三阶张量,这是一组2D全息图。将三阶张量分解为核心张量和三因子矩阵,仅使用无噪声主分量将其近似为低阶张量。通过假设半导体样品中的p-n结,将该技术应用于模拟全息图。使用张量分解,全息图和重建的相位图的峰值信噪比得到了显著改善。此外,考虑到条纹对比度和条纹相对于样品的漂移不均匀,该方法被应用于时间分辨原位电子全息术的更实际的情况。对重建的相位图的准确性和精度进行了定量评估,以证明其在束敏感材料的原位实验和低剂量实验中的有效性。
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引用次数: 4
Effect of nitrogen-containing bisphosphonates on osteoclasts and osteoclastogenesis: an ultrastructural study 含氮双磷酸盐对破骨细胞和破骨细胞生成的影响:超微结构研究
IF 1.8 4区 工程技术 Pub Date : 2021-06-01 DOI: 10.1093/jmicro/dfaa073
Yoshitoki Takagi;Satoshi Inoue;Kaoru Fujikawa;Miwako Matsuki-Fukushima;Mitsuori Mayahara;Kayo Matsuyama;Yasuo Endo;Masanori Nakamura
We have previously indicated that a single injection of alendronate, one of the nitrogen-containing bisphosphonates (NBPs), affects murine hematopoietic processes, such as the shift of erythropoiesis from bone marrow (BM) to spleen, disappearance of BM-resident macrophages, the increase of granulopoiesis in BM and an increase in the number of osteoclasts. NBPs induce apoptosis and the formation of giant osteoclasts in vitro and/or in patients undergoing long-term NBP treatment. Therefore, the time-kinetic effect of NBPs on osteoclasts needs to be clarified. In this study, we examined the effect of alendronate on mouse osteoclasts and osteoclastogenesis. One day after the treatment, osteoclasts lost the clear zone and ruffled borders, and the cell size decreased. After 2 days, the cytoplasm of osteoclasts became electron dense and the nuclei became pyknotic. Some of the cells had fragmented nuclei. After 4 days, osteoclasts had euchromatic nuclei attached to the bone surface. Osteoclasts had no clear zones or ruffled borders. After 7 days, osteoclasts formed giant osteoclasts via the fusion of multinuclear and mononuclear osteoclasts. These results indicate that NBPs affect osteoclasts and osteoclastogenesis via two different mechanisms.
我们之前已经表明,单次注射阿仑膦酸盐,一种含氮的二磷酸盐(NBPs),会影响小鼠的造血过程,如红细胞生成从骨髓(BM)转移到脾脏,骨髓固有巨噬细胞消失,骨髓中粒细胞增多和破骨细胞数量增加。NBP在体外和/或在接受长期NBP治疗的患者中诱导细胞凋亡和巨大破骨细胞的形成。因此,NBPs对破骨细胞的时间动力学效应需要澄清。在本研究中,我们检测了阿仑膦酸盐对小鼠破骨细胞和破骨细胞生成的影响。治疗一天后,破骨细胞失去了清晰的区域和褶皱的边界,细胞大小减小。2天后,破骨细胞的细胞质变得电子致密,细胞核变得致密。一些细胞的细胞核碎裂。4天后,破骨细胞具有附着在骨表面的常染色细胞核。破骨细胞没有清晰的区域或褶皱的边界。7天后,破骨细胞通过多核和单核破骨细胞的融合形成巨大的破骨细胞。这些结果表明NBPs通过两种不同的机制影响破骨细胞和破骨细胞生成。
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引用次数: 4
Performance of a silicon-on-insulator direct electron detector in a low-voltage transmission electron microscope 低压透射电子显微镜中绝缘体上硅直接电子探测器的性能
IF 1.8 4区 工程技术 Pub Date : 2021-06-01 DOI: 10.1093/jmicro/dfaa072
Takafumi Ishida;Akira Shinozaki;Makoto Kuwahara;Toshinobu Miyoshi;Koh Saitoh;Yasuo Arai
The performance of a direct electron detector using silicon-on-insulator (SOI) technology in a low-voltage transmission electron microscope (LVTEM) is evaluated. The modulation transfer function and detective quantum efficiency of the detector are measured under backside illumination. The SOI-type detector is demonstrated to have high sensitivity and high efficiency for the direct detection of low-energy electrons. The detector is thus considered suitable for low-dose imaging in an LVTEM.
在低压透射电子显微镜(LVTEM)中评估了使用绝缘体上硅(SOI)技术的直接电子探测器的性能。在背光源照射下测量了探测器的调制传递函数和探测量子效率。SOI型探测器被证明对于低能量电子的直接探测具有高灵敏度和高效率。因此,该检测器被认为适合于LVTEM中的低剂量成像。
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引用次数: 1
Introduction to EBSD: Current Issues in Data Presentation. EBSD简介:数据表示中的当前问题。
IF 1.8 4区 工程技术 Pub Date : 2021-05-01 DOI: 10.2172/1870763
JosephTan Michael
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引用次数: 0
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