MK 和要求执行手工操作和 LB 准备的图纸和制作的电影。问写的手稿, LB 写的剧本和 MK 准备的表。SM、RB 和 SP 认真阅读了表、剧本和手稿。作者感谢汉娜 Chomontowska 为 ultramicrotomy 以及叔父 Suski 和亨瑞 Bilski 提供了出色的协助, 他们对 TEM EDX 分析提供了出色的帮助。作者要感谢帕特里克林博士的专业英语语言更正和芭芭拉 Sobiak 记录的指示。
这项工作得到波兰科学和高等教育部赠款 N N401 140639 的支持, 由国家科学中心提供赠款, 波兰 2016/23/n/NZ4/03313 至磅和 2016/23/n/NZ1/02449 至 MK, 欧盟 FP7 项目 BIOIMAGINE: 生物成像在研究创新和教育, GA 264173, 和由 Nencki 实验生物学研究所的法定资金, 波兰科学院。

1. 细胞培养和治疗
<ol><li>将所有必要的材料放在层流罩下, 并在紫外线照射下消毒。文化媒介是: 1:1 混合火腿的 F12 和 DMEM 培养基与2.5 毫米 l-谷氨酰胺补充100的 u/毫升青霉素, 100 U/毫升链霉素, 0.3 毫克/毫升 G418 和10% 胎牛血清 (v/v) 为人类胎儿 hFOB 1.19 SV40 大 T 抗原转染成骨细胞, 和麦考伊的5A 培养基与1.5 毫米 l-谷氨酰胺补充 100 u/毫升青霉素, 100 u/毫升链霉素和15% 血清 (v/v) 的人骨肉瘤 Saos-2 细胞。</li>
	<li>文化 hFOB 1.19 个细胞在34°c 在 5% co2和 Saos-2 细胞的大气在37°c 在 5% co 2 的大气中.转移细胞培养, 无论是 hFOB 1.19 或 Saos-2 细胞, 从孵化器到层流罩, 并改变培养基到10毫升的新鲜培养基与血清。</li>
	<li>孵化细胞无刺激 (休息细胞) 或刺激他们与50µg/毫升抗坏血酸 (AA), 其次是7.5 毫米β-甘油 (β GP), 早在超纯水制备和过滤通过0.22 µm 注射器过滤器。将刺激从塑料离心管添加到培养基表面。轻轻搅动培养皿, 孵育7天。</li>
</ol>2. 钙矿物的检测
<ol><li>用磷酸盐缓冲盐水冲洗细胞培养物 (PBS: 125 毫米氯化钠, 5 毫米氯化钾, 10 毫米 Na2HPO4, 1 毫米,2 PO4, pH 7.0)。</li>
	<li>在 PBS 中加入5毫升的 2% (g:100 毫升) AR, pH 5.0, 并孵化出30分钟的板材以染色矿物质。</li>
	<li>用 PBS 洗3次。小心地将 PBS 添加到碟壁上, 尽量不要破坏矿物质。</li>
	<li>在倒置光显微镜下观察钙矿物, 并拍摄图像。</li>
</ol>3. 紫外光下探头的可视化
<ol><li>对于细胞裂解物, 治疗细胞培养, 无论是休息或刺激7天, 根据胶原酶消化协议24。</li>
	<li>从细胞培养中收集培养基, 用 PBS 冲洗细胞。</li>
	<li>在0.25 米蔗糖、0.12 米氯化钠、0.01 米氯化钾和0.02 米三盐酸缓冲液中, 用3毫升 500 U/毫升胶原酶的细胞消化, pH 7.45, 在37摄氏度为 3 h。</li>
	<li>机械刮细胞, 转移到塑料离心管, 并通过10次1毫升 40 U 注射器与 0.5 x 16 针。</li>
	<li>离心样品在 500 x g 5 分钟。</li>
	<li>在500µL 合成软骨淋巴中丢弃上清和悬浮细胞颗粒 (SCL, 100 毫米氯化钠, 12.7 毫米氯化钾, 0.57 毫米氯化镁2, 1.83 毫米 NaHCO3, 0.57 毫米 NaH2PO4, 5.5 毫米 d-葡萄糖, 63 毫米蔗糖, 16.5 毫米 Hepes, pH 7.4)。</li>
	<li>将羟基磷灰石 (HA)、氟磷灰石 (FA) 和 chlorapatite (CA) 粉从 UV transilluminator 上的瓶子中转移到刮刀上, 并作为控制剂使用。</li>
	<li>将细胞裂解物从塑料管中转移到塑料尖上, 并小心地放在 UV transilluminator 上。</li>
	<li>在可见光和紫外线照射下拍摄图像。</li>
</ol>4. EDX 探针的制备
<ol><li>用于阴性染色的矿物质的制备<ol>
<li>悬浮2.5 毫克综合生产 HA, CA 和 FA 矿物25在500µL 的去离子水和孵化在37°c 在一个大气 5% CO2为 1 h。</li>
		<li>将 Formvar/碳300网格镍网从盒中带抗静电钳, 放置在瓷质多孔板上, 在网格上放置10µL 的 HA、CA 和 FA 悬浮液。</li>
		<li>在室温下将样品干燥30分钟。</li>
	</ol>
</li>
	<li>嵌入26的静止和受激细胞的制备<ol>
<li>从细胞培养中收集培养基, 用生理脱敏 (PD) 培养基 (125 毫米氯化钠, 5 毫米氯化钾, 10 毫米 NaHCO3, 1 毫米的 HEPES,2 厘米, 4 毫米葡萄糖, 10 毫米, pH 20) 冲洗细胞。</li>
		<li>固定细胞与5毫升的混合物 3% (g:100 毫升) paraformaldehyde/1% (g:100 毫升) 戊二醛在100毫米磷酸钠缓冲, pH 7.2, 为 1 h 在室温下的通风罩。</li>
		<li>用5毫升100毫米磷酸钠缓冲液冲洗细胞, 洗涤后轻轻取出缓冲液。</li>
		<li>在暗室中, 将样品与2毫升 1% (g:100 毫升) 锇毒气在100毫米磷酸钠缓冲液中, pH 为 7.2, 在室温下为20分钟的烟罩。</li>
		<li>除去锇毒气并利用它。</li>
		<li>用5毫升100毫米磷酸钠缓冲液冲洗细胞。</li>
		<li>然后, 在室温下, 在5毫升的乙醇溶液系列的整除数中脱水样品: 25% (按卷) 为5分钟, 50% (按卷) 为10分钟, 75% (按卷) 为15分钟, 90% (按卷) 为20分钟。最后使用绝对乙醇两次, 孵育30分钟和12小时。</li>
		<li>机械刮伤细胞从塑料培养皿, 收集成塑料离心管和离心机的样品在 130 x g 1 分钟。</li>
		<li>将 LR 白色树脂和绝对乙醇混合物的1毫升中的细胞上清液, 并将其悬浮在1:2 的体积比上。</li>
		<li>在使用前混合好玻璃管的含量, 室温下孵育30分钟。</li>
		<li>离心样品在 130 x g 1 分钟。</li>
		<li>去除上清液, 重复前一步骤使用1毫升的1:1 混合物的 LR 白色树脂和绝对乙醇。</li>
		<li>在室温下拌匀, 孵育30分钟。</li>
		<li>离心样品在 130 x g 1 分钟。</li>
		<li>删除上清液。</li>
		<li>最后, 将1毫升纯 LR 白树脂加入样品两次, 在室温下在塑料管中孵育1小时。</li>
		<li>将每个样品的500µL 到明胶胶囊中。 注: 样品用一小张纸和一支铅笔标记, 这样树脂就不会破坏标签。</li>
		<li>关闭明胶胶囊, 放入塑料离心管和离心机, 在 130 x g 1 分钟的摆动转子。</li>
		<li>使用真空泵从塑料管上取出胶囊。</li>
		<li>将样品移动到烤箱, 聚合56摄氏度, 48 小时。</li>
		<li>将这些块装入支架并将它们修剪到金字塔上。</li>
		<li>将持有者放入 ultramicrotome 中, 附着金刚石超45°刀, 并用去离子水填充;记得要清洁刀片的附带气泡。</li>
		<li>然后切割部分 (700 Å) 使用钻石刀到去离子水浴。</li>
		<li>使用牛睫毛设置废料, 并将它们放在 Formvar/碳300网状镍网的光亮面上晾干。</li>
		<li>在黑烟罩下, 用绝对乙醇制备 2.5% (按卷) 醋酸铀。把醋酸铀放在铅容器里, 记住不要捡起沉淀物。</li>
		<li>在黑暗的房间里, counterstain 了合成磷灰石和细胞样本的网格, 用 2.5% (由卷) 醋酸铀在乙醇中的20分钟室温下在通风罩下。</li>
		<li>用50% 乙醇 (按卷) 清洗网格, 然后在去离子水中, 室温干燥24小时。最后, 把网格放进盒子里。</li>
	</ol>
</li>
</ol>5. TEM-EDX 分析
<ol><li>透射电子显微镜 (tem) 配全量程能量色散 X 射线显微分析 (EDX) 系统和11像素摄像头的 tem 成像<ol>
<li>准备一个铍持有人观察矿物和细胞。使用抗静电工具。</li>
		<li>卸下双螺钉, 将铍板和铍垫圈从固定器的其余部分提起。</li>
		<li>登上网格, 闪亮的一面, 在持有人。</li>
		<li>小心地放置铍垫圈和铍板, 拧紧螺钉。</li>
		<li>把支架放进真空室, 打开真空泵。</li>
		<li>一旦真空达到, 轻轻地将支架插入成像腔并打开光束。</li>
		<li>在荧光显示器上, 设置显微镜的孔径参数。执行图像散光校正;设置缩放、焦点和框架;并采取透射电镜图像放大50,000X。</li>
	</ol>
</li>
	<li>用于光谱和成分分析的茎显像和 X 射线微透析<ol>
<li>将能量色散 X 射线 (EDX) 探测器插入扫描透射电镜 (脑干) 成像室。</li>
		<li>调整焦点模式中图像的锐度。</li>
		<li>以15,000X 的放大倍数拍摄茎图。</li>
		<li>在样本中选择一个点, 用于 X 射线微量分析和收集光谱。</li>
		<li>通过对样本中周期表中所有元素的所有原子权重求和 (如 100%) 来获取数据, 并表示所选元素的内容: Ca、F、Cl 和 P (如 atomic%)。然后, 计算每个样品的钙、F 或 Cl 对 P 的比值。</li>
	</ol>
</li>
	<li>离子映射<ol>
<li>选取钙、氟、氯、磷等元素进行离子映射, 并对样品中所选元素进行 EDX 映射。</li>
		<li>通过指示分析元素的本地化: ca、f、Cl 和 P (作为原子%) 来获取数据, 并计算每个样本的 ca、f 或 Cl 的协定位 (%)。</li>
	</ol>
</li>
</ol>

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作者声明他们没有利益冲突。

在本文中, 我们描述了 AR 染色的协议, 氟磷灰石的紫外光识别和透射电镜-EDX 的体外成像中的矿化细胞和磁气产生的矿物质.通过遵循一些常见的故障排除步骤, 可以解决上述所有方法。为了获得最佳结果, 应该仔细地执行几个关键步骤。首先, 最好添加 AA (酸性), 其次是β GP (碱性), 以保持培养基的 pH 值7.4。其次, 经过 AR 染色后, 染色的钙沉积物非常脆弱, 细胞应小心清洗, 以防止在添加 PBS 时破坏晶体。三是始终将醋酸铀浓缩在铅容器中, 不要捡起沉淀物, 因为只有稀释的馏分才应用于固定。第四, 请记住, AA 和醋酸铀是轻敏感的, 应该在黑暗的房间处理。第五是将 LR 白与绝对乙醇混合后, 再加入样品。第六、在明胶胶囊中的样品应使用一小张纸和一支铅笔标记, 这样树脂就不会破坏标签。第七, 钻石刀的刀片应小心地从任何气泡清洗。第八, 样品应谨慎放置在 Formvar/碳膜所在的网格的光亮面上。正确定位在 TEM 图像上进行 X 射线微量分析和离子映射的点, 以限制磷灰石识别的可能问题。
虽然所提出的协议在我们的实验设计中证明是最有利的, 但可以修改它以适应不同的目标。与任何其他技术一样, 这一方法也有其局限性。其他的技术, 如 FTIR, 应用于确认或验证已被检查的元素8,12,26的获得比率的存在。本文提出的 TEM-EDX 方法是对现有方法的显著改进, 它是基于显微拉曼的映射和化学计量学方法的结合, 与银纳米粒子 (AgNPs) 分布的成像类似于各种矿物的表面和他们的分子相互作用的机制21。在两种矿物模型 (宏观和微观尺度) 上, 对拉曼成像进行了测试。用韦斯普奇软件对每种样品/控制的拉曼光谱进行了分析, 并通过 ICP、AFM 和扫描电镜 EDX 等方法对结果进行了验证。本文还通过显微分析套件软件对30帧和n = 6 光谱进行了 EDX 映射, 并通过 FTIR、调频、TEM、SPM 和3D 地形 AFM4等方法对其结果进行了验证。,5. 拟议的 TEM-EDX 微分析方法, 如拉曼成像技术, 只需要最起码的样品制备, 是超敏感、无侵入性和成本效益的, 并可扩展到与人类环境有关的其他系统。
所提出的技术可能会在未来广泛的研究领域中使用。可对不同的天然和合成磷灰石进行改造。它也可以调整, 以适应生物和化学探针实验的协议。此外, 可以对周期表中的所有元素进行分析, 以便不仅可视化矿物的形态, 还能直观地看到它们的离子成分和替代物的变化。离子交换在磷灰石是一个已知的程序为生物医学应用27。一个可能的生理因素, 控制钙, F 和 Cl 离子或锶, 镁, 锰和锌金属离子置换在形成过程中的 HA, 这是一个转折点之间的生理和病理成矿, 可能是 Pi/PP我比率7。如果应用得当, 这种技术允许连续数次对同一细胞、水泡或矿物质区域的多个元素进行映射。
近年来, 骨矿化机制28、29、30、31、32、33等方面取得了很大进展。我们认识到, 不同的细胞系可能有不同的形状的囊泡和矿物质形成。在这方面, 我们选择了两个人类细胞系: 成骨 hFOB 1.19 和骨肉瘤 Saos-2, 它经历了从增殖到成矿的完全成骨分化, 并产生了在 ECM 34中触发磷灰石成核的促动,35。
钙矿床的 AR 染色表明, 受刺激的 Saos-2 细胞矿化程度不同于 hFOB 1.19 成骨细胞。与 hFOB 1.19 细胞产生的 Saos-2 细胞相比, 这与矿物的产生有关。EDX 数据显示, 由于钙和磷分布的重叠, Saos-2 细胞囊泡产生的矿物质与合成 HA 相似。与此相反, hFOB 1.19 细胞囊泡中氟和磷分布的重叠表明, 除磷灰石外, 其他类型矿物的形成。
最后, 我们的发现表明, 囊泡是矿物成核的关键决定因素, 特别是在细胞级5,25。此外, 我们的数据与早先的理论一致, 即可将微泡作为一种专门的形式, 能够在细胞4的外部开始矿化。Saos-2 细胞释放出的囊泡的比较, 矿化效率更高, hFOB 1.19 细胞释放出的囊泡, 采用 TEM-EDX microanalytical 方法, 支持了一种假设, 即 "磁囊" 是矿物填充泡。这就引出了一个问题, 那就是是否需要有 mv 的存在来诱导磷灰石成核, 以及这如何改变生理对病理成矿作用。

骨是由两部分组成的结缔组织: 有机 (细胞和胶原纤维) 和矿物质 (钙和磷酸盐化合物)。骨中主要矿物成分为磷灰石1。不同类型的成矿能力的细胞在骨 (成骨细胞), 在牙齿 (成牙本质细胞) 和软骨 (软骨细胞) 通过产生细胞外基质 (ECM) 和释放基质的蛋白质来调节矿化的初始步骤囊泡 (mv) (图 1)。100-300 毫微米直径囊泡, 积累钙和磷酸促进磷灰石成核, 随后绑定到胶原蛋白2,3。然后, 磷灰石释放到胞外培养基中。磷灰石继续与胶原纤维接触, 形成骨基质。矿化由 Pi和钙2 +在胞外培养基中的恒定供给维持。最近发布的一些数据支持我们的模型4,5。软组织在生理条件下不矿化。然而, 异位钙化可能发生在病理条件下, 如血管钙化3。获得成骨细胞表型的血管细胞可产生诱导磷灰石的核化, 并在血管壁内侧和内膜层启动成矿作用。由于异位钙化类似于正常的软骨矿化3, 了解骨细胞和软骨细胞成矿作用的分子机制, 应为软组织异位钙化提供一些线索。形成。
骨骼组织的发育受各种酶、生长因子、成矿作用的促进剂或抑制剂的调节。组织非特异性碱性磷酸酶 (TNAP) 的拮抗作用 (图 1) 和 ectonucleotide 焦磷酸酶/磷酸二酯化 I (NPP1), 连同锚蛋白 (谢谢), 控制无机焦磷酸盐 (PPI) 浓度6. PPi, 一种有效的 HA 形成抑制剂, 由 TNAP 水解;NPP1 水解核苷酸 triphosphates 形成 ppi , 而谢谢出口 ppi从细胞到 ECM。Pi/PPi 比率可能调控磷灰石形成7,8以可能的病理后果9。
MV 膜富含离子传输蛋白, 在核过程中促进了钙和磷酸盐的初始沉淀 (图 1)。磷酸盐传送器 1 (坑) 帮助合并 Pi生成在 perivesicular 空间入10,11。Annexins 可能涉及 Ca2 +的结合和运输, 以及在 MV 腔12,13中启动矿化的生物物理过程中。我们赞成早些时候提出的假说, 即在其在 ECM14、15中传播之前, 在 MV 内的磷灰石内部成核的胞质囊泡中的矿化。体外建模证实了从 PS 和 AnxA516的 proteoliposomes 中诱导的钙2 +/Pi复合物的形成。这可能表明, 钙2 +, Pi, AnxA5 和 PS 配合物在微绒毛状的 membranesrepresent 中的核核 (NC) 在 M 的 Annexins 和 TNAP也具有胶原结合可能有助于在胶原纤维上放置 mv, 并在促进电解质中的矿化传播方面的能力。牛胎球蛋白 A 和骨桥蛋白 (OPN)17, 被称为磷灰石形成抑制剂, 可能减慢矿化在胶原支架上的传播。成核和传播是不同的事件, 前者前, 两者可能与病理成矿过程有关。
为了发现磷酸钙络合物的化学物质如何改变生理成矿作用和异位钙化, 有必要确定细胞产生的矿物质。磷灰石是一组含有矿物质的钙和磷酸盐, 一般晶体单元细胞配方钙10(PO4)6X2, 其中 X = Cl, F, OH。分类如下18: 氟磷灰石 (FA) 钙10(PO4)6F2, chlorapatite (ca) Ca10(PO4)6Cl2和羟基磷灰石 (HA) 钙10(po4)6(OH)2。
由于每一个细胞系都呈现出明显的矿化特征, 因此选择成骨细胞系诱导矿物形成是至关重要的。在这份报告中, 我们比较了两个选定的人体细胞模型的矿物成核: 成骨 hFOB 1.19 细胞和骨肉瘤 Saos-2 细胞。骨肉瘤衍生细胞通常被用作成骨模型, Saos-2 细胞保存了最成熟的成骨19 , 而未分化的人胎儿 hFOB 细胞作为正常成骨模型被广泛使用。分化20。用不同的方法分析了它们的成矿剖面: 茜素红 s (AR) 染色、紫外 (紫外) 光可视化、透射电镜 (TEM) 成像、能量色散 X 射线微分析 (EDX) 定量和离子映射。EDX 在以往研究中使用的替代技术的优势在于, 它给出了4、5、21磷灰石晶体中离子置换的定量和定性结果。利用 TEM EDX 的总体目标是在成矿过程的不同阶段, 寻找一种简单的方法, 用于成像和定量的各种矿物中的钙、F 和 Cl 离子的分布。该方法成功地用于监测锌纳米粒子与共存化学物质的相互作用及其对水生生物的联合作用22。在另一项研究中, 用电感耦合等离子体发射光谱 (ICP)、N2 储 (注)、XRD、紫外-比 DRS、红外光谱、拉曼光谱等方法, 对钛材料在水溶液中的铜光催化剂进行了广泛的表征。光谱学, 透射电镜 EDX 和光电测量23。我们的目的是比较两个细胞系中的囊泡和矿物质的来源和性质, 以了解在骨分化过程中控制成矿作用的机制。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57423/57423fig1.jpg"> 图 1.从膜中合成细胞外基质 (ECM) 蛋白和释放基质泡 (mv) 的骨细胞中初始化的步骤方案。通过钙结合蛋白、annexins 和磷酸酯的作用, 通过无机磷酸盐转运体 (坑) 的作用, 通过组织非特异碱性磷酸酶 (TNAP) 的活性来积累钙, dephosphorylatesPPi对 Pi, 从而促进磷灰石成核。然后, 磷灰石分解并释放出细胞外培养基。矿化由 Pi和 Ca2 +在胞外介质4,5的恒定供应维持。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57423/57423fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#8217;s DMEM/F12 media mixture</td>
			<td>PAA</td>
			<td>E15-813</td>
			<td>1:1, for human fetus hFOB 1.19 SV40 large T antigen transfected osteoblasts (ATCC CRL-11372)</td>
		</tr>
		<tr>
			<td>McCoy&#8217;s 5A medium</td>
			<td>PAA</td>
			<td>E82312-0025</td>
			<td>for human osteosarcoma Saos-2 cells (ATCC HTB-85)</td>
		</tr>
		<tr>
			<td>Antibiotics mixture (penicillin/streptomycin)</td>
			<td>Sigma</td>
			<td>P0781-100ML</td>
			<td>100 U/mL each</td>
		</tr>
		<tr>
			<td>G-418</td>
			<td>Sigma</td>
			<td>68168</td>
			<td>0.3 mg/mL</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>10270</td>
			<td>10% for hFOB 1.19 and 15% for Saos-2</td>
		</tr>
		<tr>
			<td>AA</td>
			<td>Sigma</td>
			<td>A-5960</td>
			<td>50 &#181;g/mL</td>
		</tr>
		<tr>
			<td>&#223;-GP</td>
			<td>Sigma</td>
			<td>G9422-100G</td>
			<td>7.5 mM</td>
		</tr>
		<tr>
			<td>Bio-Gel HTP Gel</td>
			<td>Bio-Rad</td>
			<td>130-0420</td>
			<td>for HA</td>
		</tr>
		<tr>
			<td>FA</td>
			<td></td>
			<td></td>
			<td>synthesized by us</td>
		</tr>
		<tr>
			<td>CA</td>
			<td></td>
			<td></td>
			<td>synthesized by us</td>
		</tr>
		<tr>
			<td>Sodium phosphate buffer Na2HPO4/NaH2PO4 mixture</td>
			<td>Sigma</td>
			<td>S7907/S8282</td>
			<td>0.1 M, pH 7.2</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td></td>
			<td></td>
			<td>pH 7.0, prepared by us</td>
		</tr>
		<tr>
			<td>AR-S in PBS</td>
			<td>Sigma</td>
			<td>A5533-25G</td>
			<td>0.5 g/100 mL, pH 5.0</td>
		</tr>
		<tr>
			<td>Collagenase type IA</td>
			<td>Sigma</td>
			<td>C2674</td>
			<td>500 U/mL</td>
		</tr>
		<tr>
			<td>SCL buffer</td>
			<td></td>
			<td></td>
			<td>prepared by us</td>
		</tr>
		<tr>
			<td>Deionized wather</td>
			<td></td>
			<td></td>
			<td>produced by us</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>POCh</td>
			<td>BA6480111</td>
			<td>absolut 99.8% and solutions 25, 50, 75, 90%</td>
		</tr>
		<tr>
			<td>Uranyl acetate in 50% ethanol</td>
			<td>Polysciences Inc.</td>
			<td>21447-25</td>
			<td>0.25 g/10 mL</td>
		</tr>
		<tr>
			<td>PD medium</td>
			<td></td>
			<td></td>
			<td>pH 7.4, prepared by us</td>
		</tr>
		<tr>
			<td>Fixation mixture (paraformaldehyde/glutaraldehyde)</td>
			<td>Sigma</td>
			<td>158127/G-6257</td>
			<td>3%:1%</td>
		</tr>
		<tr>
			<td>Post-fixation OsO4</td>
			<td>Sigma</td>
			<td>75633</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>LR White resin in ethanol</td>
			<td>Polysciences Inc.</td>
			<td>17411-MUNC 500g</td>
			<td>1:2, 1:1, 100%</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>CHEMPUR</td>
			<td>111024800</td>
			<td>pure</td>
		</tr>
		<tr>
			<td>Tool</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cryogenic vials</td>
			<td>Corning Inc.</td>
			<td>430487</td>
			<td>1.2 mL</td>
		</tr>
		<tr>
			<td>Plastic Petri culture dishes</td>
			<td>Falcon</td>
			<td>353003</td>
			<td>100 mm</td>
		</tr>
		<tr>
			<td>Plastic tubes</td>
			<td>Falcon</td>
			<td>352096 and 352070</td>
			<td>15 and 50 mL</td>
		</tr>
		<tr>
			<td>Serological pipettes</td>
			<td>Falcon and VWR</td>
			<td>357521 and 612-3700</td>
			<td>1 and 10 mL</td>
		</tr>
		<tr>
			<td>Plastic microcentrifuge tubes</td>
			<td>Sigma</td>
			<td>Z688312 and Z628034</td>
			<td>1.5 mL black and 2 mL transparent</td>
		</tr>
		<tr>
			<td>Plastic tips</td>
			<td>VWR</td>
			<td>613-0364, 613-0239 and 613-1050</td>
			<td>0.1-10 &#181;L natural, 1-200 &#181;L yellow and 200-1000 &#181;L blue</td>
		</tr>
		<tr>
			<td>Plastic racks</td>
			<td>Light Labs</td>
			<td>A-7055-Z, A-7053-C</td>
			<td>green for tubes, orange for micro tubes and blue for TEM probes</td>
		</tr>
		<tr>
			<td>Laminar Hera Save</td>
			<td>Thermo Scientific Co.</td>
			<td>KS12</td>
			<td>HEPA filter (H14 according to DIN EN 1822)</td>
		</tr>
		<tr>
			<td>Incubators Hera Cell</td>
			<td>Thermo Scientific Co.</td>
			<td>150</td>
			<td>34&#176;C for hFOB 1.19 and 37&#176;C for Saos-2</td>
		</tr>
		<tr>
			<td>Fume hood</td>
			<td>POLON</td>
			<td>WCS-2</td>
			<td>for TEM stainings</td>
		</tr>
		<tr>
			<td>Glass bottles</td>
			<td>SIMAX</td>
			<td>1632414501050 and 1632414501100</td>
			<td>50 and 100 mL</td>
		</tr>
		<tr>
			<td>Quartz glass tubes</td>
			<td>SIMAX</td>
			<td>638422010100</td>
			<td>&#216; 10 mm, L 100 mm</td>
		</tr>
		<tr>
			<td>Pump</td>
			<td>IBS Integra Biosciences</td>
			<td>VACUSAFE comfort</td>
			<td>for vacuum</td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>Memmert</td>
			<td>UNE 400</td>
			<td>56&#176;C</td>
		</tr>
		<tr>
			<td>Porcelain multi-well plate</td>
			<td>Rosenthal technik</td>
			<td>229/12</td>
			<td>12 wells</td>
		</tr>
		<tr>
			<td>Glass beakers</td>
			<td>SIMAX</td>
			<td>632417010025</td>
			<td>25 mL</td>
		</tr>
		<tr>
			<td>Glass bottles</td>
			<td>Pocord</td>
			<td>DIN22</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>Plastic box</td>
			<td>Agar Scientific Ltd.</td>
			<td></td>
			<td>for darkness</td>
		</tr>
		<tr>
			<td>Snap Fit Gelatin Capsules</td>
			<td>Agar Scientific Ltd.</td>
			<td>G3741</td>
			<td>size 1</td>
		</tr>
		<tr>
			<td>Formvar/Carbon 300 Mesh Ni grids in box</td>
			<td>Agar Scientific Ltd.</td>
			<td>S162N3</td>
			<td>film on the shiny side</td>
		</tr>
		<tr>
			<td>Silicon cell scraper</td>
			<td>Sigma</td>
			<td>SIAL0010-100EA</td>
			<td>size 1.8/25 cm</td>
		</tr>
		<tr>
			<td>Syringe with needle</td>
			<td>BogMark</td>
			<td>007</td>
			<td>syringe 1 mL 40 U, needle 0.5 x 16</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Chirana</td>
			<td>CH005L</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>MPW Medical Instruments</td>
			<td>MPW-350R</td>
			<td>130 x g and 500 x g</td>
		</tr>
		<tr>
			<td>UV transluminator</td>
			<td>UVP</td>
			<td>M-20</td>
			<td>for visible and UV light</td>
		</tr>
		<tr>
			<td>Ultramicrotome</td>
			<td>LKB</td>
			<td>NOVA</td>
			<td>700&#197; sections</td>
		</tr>
		<tr>
			<td>Block holder</td>
			<td>LKB</td>
			<td>E6711</td>
			<td>round shape</td>
		</tr>
		<tr>
			<td>Diamond knife</td>
			<td>DiATOME</td>
			<td>Ultra 45&#176;</td>
			<td>size 3</td>
		</tr>
		<tr>
			<td>Eyelash holder</td>
			<td></td>
			<td></td>
			<td>bovine, prepared by us</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>ROTH</td>
			<td>2855.1</td>
			<td>antistatic for grids</td>
		</tr>
		<tr>
			<td>Spatulas set</td>
			<td>ROTH</td>
			<td>E286.1</td>
			<td>antistatic for powders</td>
		</tr>
		<tr>
			<td>Imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Light Microscope</td>
			<td>Zeiss with Canon</td>
			<td>AxioObserver Z1 equipped with PowerShot G9</td>
			<td>Phase contrast, Transmitted light, 20 x objective, RGB filters</td>
		</tr>
		<tr>
			<td>Transmission Electron Microscope</td>
			<td>TEM Jeol Co. with Oxford Instruments and SiS-Olympus</td>
			<td>JEM-1400 TEM equipped with full range INCA Energy Dispersive X-ray microanalysis (EDX) System and 11 Megapixel MORADA G2 camera</td>
			<td>magnification 50,000X for TEM and 15,000X for STEM and EDX</td>
		</tr>
		<tr>
			<td>Camera body and lenses</td>
			<td>Nikon</td>
			<td>Nikon D7100 
			Nikkor AF Micro 105 mm f/2.8D 
			Nikkor AF-S 50 mm f/1.8G 
			Nikkor AF 28 mm f/2.8D
</td>
			<td>for movie recordings</td>
		</tr>
		<tr>
			<td>Microphone</td>
			<td>MXL Mics</td>
			<td>Tempo</td>
			<td>for voice recordings</td>
		</tr>
	</tbody>
</table>

analysis of minerals produced by hfob 1.19 and saos-2 cells using transmission electron microscopy with energy dispersive x-ray microanalysis

本视频介绍了利用透射电镜与能量色散 X 射线微分析 (TEM-EDX) 比较的矿物质在囊泡释放的两个人骨细胞线: hFOB 1.19 和 Saos-2。这些细胞系, 经抗坏血酸 (AA) 和β甘油 (β GP) 治疗后, 经过完全成骨分化, 从增殖到成矿, 产生基质泡 (mv), 触发磷灰石成核在细胞外基质 (ECM)。
基于茜素红 s (AR) 染色, 利用透射电镜成像和 EDX 定量和离子图谱对细胞裂解物中的矿物质组成进行分析, 并对其进行了研究, 得出了 Saos-2 和成骨肉瘤hFOB 1.19 细胞显示出明显的矿化剖面。Saos-2 细胞比 hFOB 1.19 细胞矿化更有效, 并产生较大的矿床, 在紫外光下不可见, 但与羟基磷灰石 (HA) 相似, 因为它们有更多的钙和 F 替代物。
利用这些技术得到的结果使我们得出结论, 矿化过程的不同取决于细胞类型。我们建议, 在细胞层面上, 囊泡的起源和性质预先确定了矿物质的类型。

EDX 允许在体外成像的基质囊泡 (mv) 释放的矿化细胞和矿物生产的 MVs.利用该技术得到的结果表明, 矿化过程可能会有不同的进展。在不同类型的细胞中。两个细胞系接受相同的成骨细胞转分化治疗, 但刺激 Saos-2 单元矿化更有效地比 hFOB 1.19 成骨细胞, 由 AR 染色证明 (图 2)。这可能是由于更成熟的成骨细胞表型的 Saos-2 单元19相比 hFOB 1.19 细胞20。使用 uv transilluminator 对样品进行可视化, 表明只有在紫外线照射下才能观察到 fluorapatites (图 3)。透射电镜图像表明, 受刺激的 Saos-2 细胞产生更多的含矿物质的囊泡, 与受刺激的 hFOB 细胞或静止的 Saos-2 细胞 (图 4, 左侧和中间板) 相比较。合成磷灰石有不同形式的矿物质 (图 4, 右面板)。透射电镜图像显示 hFOB 1.19 和 Saos-2 细胞在休息和刺激条件下存在囊泡 (图 5, 左面板)。在 EDX 离子地图上, 红色点表示钙, 绿色点显示磷和蓝色点指向氟分布 (图 5, 中间板)。在受刺激的条件下, Saos-2 细胞产生的囊泡中的钙和磷分布与 hFOB 1.19 细胞产生的囊泡中的氟和磷之间有很强的重叠 (图 5, 右面板)。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57423/57423fig2.jpg"> 图 2.由 hFOB 1.19 (上部板) 和 Saos-2 (下板) 细胞所产生的矿物质 (红) 染色, 无论是休息 (R) 或经过7天的刺激与 AA 和β GP (S).酒吧:25 µm。给出了一个典型的n = 3 的结果。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57423/57423fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57423/57423fig3.jpg"> 图 3.可见 (上部面板) 和 UV (中间板) 的合成 HA 的光可视化, 钙和 FA 矿物以及由 hFOB 1.19 (左下板) 和 Saos-2 (右下板) 细胞所产生的矿物质, 无论是休息 (R) 或经过7天的刺激与 AA 和β GP (S).<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57423/57423fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a> 
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57423/57423fig4.jpg"> 图 4. hFOB 1.19 (左面板) 和 Saos-2 (中间板) 细胞产生的囊泡和矿物质的透射电镜图像, 无论是静止 (R) 还是经过7天的刺激与 AA 和β GP (S)。合成 HA, CA 和 FA 矿物显示为控制 (右面板).酒吧: 白色500毫微米, 黑色250毫微米。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57423/57423fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57423/57423fig5.jpg"> 图 5. TEM 图像 (左面板) 的 hFOB 1.19 和 Saos-2 细胞要么休息 (R) 或后7天的刺激与 AA 和β GP (S)。离子地图为钙 (红色), Cl (黄色), F (蓝色) 和 P (绿色) 从 EDX (中间板)。元素的共同定位 (右面板): 钙对磷 (Ca), 氟到磷 (F), 或氯对磷 (Cl) 是根据 EDX 地图量化.酒吧: 500 毫微米。对两个细胞系进行了三项独立实验。十图片从每个变形 (休息和被刺激) 被采取了, 然后从2到6他们被选择了为元素 co 本地化的进一步计算。给出了一个典型的结果。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57423/57423fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis at JoVE.com

Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis

我们提出一个协议, 以比较的矿物质在囊泡释放的两个人骨细胞系: hFOB 1.19 和 Saos-2。利用茜素红 s (AR) 染色、紫外 (紫外) 光可视化、透射电镜 (TEM) 成像和能量色散 X 射线显微分析 (EDX) 对其成矿剖面进行了研究。

透射电镜 hFOB 1.19 和 Saos-2 细胞产生的能量色散 X 射线显微分析

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in ...

analysis-minerals-produced-hfob-119-saos-2-cells-using-transmission

Nanyang Technological University

Research

JoVE Journal

Chemistry

9.1K 次观看.  Polish Academy of Sciences. 我们提出一个协议, 以比较的矿物质在囊泡释放的两个人骨细胞系: hFOB 1.19 和 Saos-2。利用茜素红 s (AR) 染色、紫外 (紫外) 光可视化、透射电镜 (TEM) 成像和能量色散 X 射线显微分析 (EDX) 对其成矿剖面进行了研究。

视频: Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis

Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over large or small sample areas. It has been applied in many areas of science, including materials science, geoscience, studying works of cultural heritage, and in chemical biology. In the case of chemical biology, for example, visualizing the metal distributions within cells allows us to study both naturally-occurring metal ions in the cells, as well as exogenously-introduced metals such as drugs and nanoparticles. Due to the fully hydrated nature of nearly all biological samples, cryo-fixation followed by imaging under cryogenic temperature represents the ideal imaging modality currently available. However, under the circumstances that such a combination is not easily accessible or practical, aldehyde based chemical fixation remains useful and sometimes inevitable. This article describes in as much detail as possible in the preparation of adherent mammalian cells by chemical fixation for X-ray fluorescent imaging.

Here, we present a protocol on how to determine the quantity and distribution of metals in a sample using synchrotron X-ray fluorescence. We focus on adherent cells, and describe the chemical fixation method to prepare this sample. We then describe how to mount and image the sample using synchrotron X-rays.

化学固定准备贴壁细胞的X射线荧光成像

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over ...

科研

化学

1,3,5-Triphenylbenzene and Corannulene as Electron Receptors for Lithium Solvated Electron Solutions

The authors report on conductivity studies carried out on lithium solvated electron solutions (LiSES) prepared using two types of polyaromatic hydrocarbons (PAH), namely 1,3,5-triphenylbenzene and corannulene, as electron receptors. The solid PAHs were first dissolved in tetrahydrofuran (THF) to form a solution. Metallic lithium was then dissolved into these PAH/THF solutions to yield either blue or greenish blue solutions, colors which are indicative of the presence of solvated electrons. Conductivity measurements at ambient temperature carried out on 1,3,5-triphenylbenzene-based LiSES, denoted by LixTPB(THF)24.7 (x = 1, 2, 3, 4), showed an increase of conductivity with increase of Li:PAH ratio from x = 1 to 2. However, the conductivity gradually decreased upon further increasing the ratio. Indeed the conductivity of LixTPB(THF)24.7 for x = 4 is even lower than for x = 1. Such behavior is similar to that of the previously reported LiSES prepared from biphenyl and naphthalene. Conductivity versus temperature measurements on corannulene-based LiSES, denoted by LixCor(THF)247 (x = 1, 2, 3, 4, 5), showed linear relationships with negative slopes, indicating a metallic behavior similar to biphenyl and naphthalene-based LiSES.

The authors report on conductivity studies carried out on lithium solvated electron solutions (LiSES) prepared using 1,3,5-triphenylbenzene (TPB) and corannulene as electron receptors.

1,3,5-三苯和碗烯作为电子受体锂溶剂化电子解决方案

The authors report on conductivity studies carried out on lithium solvated electron solutions (LiSES) prepared using two types of polyaromatic ...

Quantifying X-Ray Fluorescence Data Using MAPS

The quantification of X-ray fluorescence (XRF) microscopy maps by fitting the raw spectra to a known standard is crucial for evaluating chemical composition and elemental distribution within a material. Synchrotron-based XRF has become an integral characterization technique for a variety of research topics, particularly due to its non-destructive nature and its high sensitivity. Today, synchrotrons can acquire fluorescence data at spatial resolutions well below a micron, allowing for the evaluation of compositional variations at the nanoscale. Through proper quantification, it is then possible to obtain an in-depth, high-resolution understanding of elemental segregation, stoichiometric relationships, and clustering behavior.
This article explains how to use the MAPS fitting software developed by Argonne National Laboratory for the quantification of full 2-D XRF maps. We use as an example results from a Cu(In,Ga)Se2 solar cell, taken at the Advanced Photon Source beamline 2-ID-D at Argonne National Laboratory. We show the standard procedure for fitting raw data, demonstrate how to evaluate the quality of a fit and present the typical outputs generated by the program. In addition, we discuss in this manuscript certain software limitations and offer suggestions for how to further correct the data to be numerically accurate and representative of spatially resolved, elemental concentrations.

Here, we demonstrate the use of the X-ray fluorescence fitting software, MAPS, created by Argonne National Laboratory for the quantification of fluorescence microscopy data. The quantified data that results is useful for understanding the elemental distribution and stoichiometric ratios within a sample of interest.

利用地图量化 X 射线荧光数据

The quantification of X-ray fluorescence (XRF) microscopy maps by fitting the raw spectra to a known standard is crucial for evaluating chemical ...

Liquid-cell Transmission Electron Microscopy for Tracking Self-assembly of Nanoparticles

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully understood. We have traced the trajectories of individual nanoparticles using liquid-cell transmission electron microscopy (TEM) to investigate the mechanism of the assembly process. Herein, we present the protocols used for liquid-cell TEM studies of the self-assembly mechanism. First, we introduce the detailed synthetic protocols used to produce uniformly sized platinum and lead selenide nanoparticles. Next, we present the microfabrication processes used to produce liquid cells with silicon nitride or silicon windows and then describe the loading and imaging procedures of the liquid-cell TEM technique. Several notes are included to provide helpful tips for the entire process, including how to manage the fragile cell windows. The individual motions of nanoparticles tracked by liquid-cell TEM revealed that changes in the solvent boundaries caused by evaporation affected the self-assembly process of nanoparticles. The solvent boundaries drove nanoparticles to primarily form amorphous aggregates, followed by flattening of the aggregates to produce a 2-dimensional (2D) self-assembled structure. These behaviors are also observed for different nanoparticle types and different liquid-cell compositions.

Here we introduce experimental protocols for the real-time observation of a self-assembly process using liquid-cell transmission electron microscopy.

用于跟踪纳米粒子自组装的液体细胞透射电子显微镜

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully ...

Obtaining 3D Chemical Maps by Energy Filtered Transmission Electron Microscopy Tomography

Energy filtered transmission electron microscopy tomography (EFTEM tomography) can provide three-dimensional (3D) chemical maps of materials at a nanometric scale. EFTEM tomography can separate chemical elements that are very difficult to distinguish using other imaging techniques. The experimental protocol described here shows how to create 3D chemical maps to understand the chemical distribution and morphology of a material. Sample preparation steps for data segmentation are presented. This protocol permits the 3D distribution analysis of chemical elements in a nanometric sample. However, it should be noted that currently, the 3D chemical maps can only be generated for samples that are not beam sensitive, since the recording of filtered images requires long exposure times to an intense electron beam. The protocol was applied to quantify the chemical distribution of the components of two different heterogeneous catalyst supports. In the first study, the chemical distribution of aluminum and titanium in titania-alumina supports was analyzed. The samples were prepared using the swing-pH method. In the second, the chemical distribution of aluminum and silicon in silica-alumina supports that were prepared using the sol-powder and mechanical mixture methods was examined.

This paper describes a protocol to achieve 3D chemical maps combining energy filtered imaging and electron tomography. The chemical distribution of two catalyst supports formed by elements that are difficult to distinguish by other imaging techniques was studied. Each application consists of mapping overlapped chemical elements - respectively spaced-ionization edges.

通过能量过滤透射电镜成像获得3D 化学图

Energy filtered transmission electron microscopy tomography (EFTEM tomography) can provide three-dimensional (3D) chemical maps of materials at a ...

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth. After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 &#181;m grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography. As almost every crystal can be used for diffraction data collection, these microfluidic chips are a very efficient crystal delivery method.

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes in detail how to fabricate and operate microfluidic devices for X-ray diffraction data collection at room temperature. Additionally, it describes how to monitor protein crystallization by dynamic light scattering and how to process and analyze obtained diffraction data.

用于原位晶体 x 射线衍射和原位的微流控芯片系列晶体学的动态光散射

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The ...

Elemental-sensitive Detection of the Chemistry in Batteries through Soft X-ray Absorption Spectroscopy and Resonant Inelastic X-ray Scattering

Energy storage has become more and more a limiting factor of today's sustainable energy applications, including electric vehicles and green electric grid based on volatile solar and wind sources. The pressing demand of developing high-performance electrochemical energy storage solutions, i.e., batteries, relies on both fundamental understanding and practical developments from both the academy and industry. The formidable challenge of developing successful battery technology stems from the different requirements for different energy-storage applications. Energy density, power, stability, safety, and cost parameters all have to be balanced in batteries to meet the requirements of different applications. Therefore, multiple battery technologies based on different materials and mechanisms need to be developed and optimized. Incisive tools that could directly probe the chemical reactions in various battery materials are becoming critical to advance the field beyond its conventional trial-and-error approach. Here, we present detailed protocols for soft X-ray absorption spectroscopy (sXAS), soft X-ray emission spectroscopy (sXES), and resonant inelastic X-ray scattering (RIXS) experiments, which are inherently elemental-sensitive probes of the transition-metal 3d and anion 2p states in battery compounds. We provide the details on the experimental techniques and demonstrations revealing the key chemical states in battery materials through these soft X-ray spectroscopy techniques.

Here, we present a protocol for typical experiments of soft X-ray absorption spectroscopy (sXAS) and resonant inelastic X-ray scattering (RIXS) with applications in battery material studies.

软 x 射线吸收光谱和共振非弹性 x 射线散射法检测电池化学成分的元素敏感度

Energy storage has become more and more a limiting factor of today's sustainable energy applications, including electric vehicles and green electric grid ...

Using Graphene Liquid Cell Transmission Electron Microscopy to Study in Situ Nanocrystal Etching

Graphene liquid cell electron microscopy provides the ability to observe nanoscale chemical transformations and dynamics as the reactions are occurring in liquid environments. This manuscript describes the process for making graphene liquid cells through the example of graphene liquid cell transmission electron microscopy (TEM) experiments of gold nanocrystal etching. The protocol for making graphene liquid cells involves coating gold, holey-carbon TEM grids with chemical vapor deposition graphene and then using those graphene-coated grids to encapsulate liquid between two graphene surfaces. These pockets of liquid, with the nanomaterial of interest, are imaged in the electron microscope to see the dynamics of the nanoscale process, in this case the oxidative etching of gold nanorods. By controlling the electron beam dose rate, which modulates the etching species in the liquid cell, the underlying mechanisms of how atoms are removed from nanocrystals to form different facets and shapes can be better understood. Graphene liquid cell TEM has the advantages of high spatial resolution, compatibility with traditional TEM holders, and low start-up costs for research groups. Current limitations include delicate sample preparation, lack of flow capability, and reliance on electron beam-generated radiolysis products to induce reactions. With further development and control, graphene liquid cell may become a ubiquitous technique in nanomaterials and biology, and is already being used to study mechanisms governing growth, etching, and self-assembly processes of nanomaterials in liquid on the single particle level.

Using Graphene Liquid Cell Transmission Electron Microscopy to Study in Situ Nanocrystal Etching

Graphene liquid cell electron microscopy can be used to observe nanocrystal dynamics in a liquid environment with greater spatial resolution than other liquid cell electron microscopy techniques. Etching premade nanocrystals and following their shape using graphene liquid cell Transmission Electron Microscopy can yield important mechanistic information about nanoparticle transformations.

用石墨烯液细胞透射电镜研究原位纳米晶蚀刻

Graphene liquid cell electron microscopy provides the ability to observe nanoscale chemical transformations and dynamics as the reactions are occurring in ...

Preparation of Graphene-Supported Microwell Liquid Cells for In Situ Transmission Electron Microscopy

The fabrication and preparation of graphene-supported microwell liquid cells (GSMLCs) for in situ electron microscopy is presented in a stepwise protocol. The versatility of the GSMLCs is demonstrated in the context of a study about etching and growth dynamics of gold nanostructures from a HAuCl4 precursor solution. GSMLCs combine the advantages of conventional silicon- and graphene-based liquid cells by offering reproducible well depths together with facile cell manufacturing and handling of the specimen under investigation. The GSMLCs are fabricated on a single silicon substrate which drastically reduces the complexity of the manufacturing process compared to two-wafer-based liquid cell designs. Here, no bonding or alignment process steps are required. Furthermore, the enclosed liquid volume can be tailored to the respective experimental requirements by simply adjusting the thickness of a silicon nitride layer. This enables a significant reduction of window bulging in the electron microscope vacuum. Finally, a state-of-the-art quantitative evaluation of single particle tracking and dendrite formation in liquid cell experiments using only open source software is presented.

Preparation of Graphene-Supported Microwell Liquid Cells for In Situ Transmission Electron Microscopy

A protocol for preparation of graphene-supported microwell liquid cells for in situ electron microscopy of gold nanocrystals from HAuCl4 precursor solution is presented. Furthermore, an analysis routine is presented to quantify observed etching and growth dynamics.

石墨烯支持的微井液体细胞制备,用于原位透射电子显微镜

The fabrication and preparation of graphene-supported microwell liquid cells (GSMLCs) for in situ electron microscopy is presented in a stepwise protocol. ...

Biological Samples Preparation for Speciation at Cryogenic Temperature using High-Resolution  X-Ray Absorption Spectroscopy

The study of elements with X-ray absorption spectroscopy (XAS) is of particular interest when studying the role of metals in biological systems. Sample preparation is a key and often complex procedure, particularly for biological samples. Although X-ray speciation techniques are widely used, no detailed protocol has been yet disseminated for users of the technique. Further, chemical state modification is of concern, and cryo-based techniques are recommended to analyze the biological samples in their near-native hydrated state to provide the maximum preservation of chemical integrity of the cells or tissues. Here, we propose a cellular preparation protocol based on cryo-preserved samples. It is demonstrated in a high energy resolution fluorescence detected X-ray absorption spectroscopy study of selenium in cancer cells and a study of iron in phytoplankton. This protocol can be used with other biological samples and other X-ray techniques that can be damaged by irradiation.

This protocol presents a detailed procedure to prepare biological cryosamples for synchrotron-based X-ray absorption spectroscopy experiments. We describe all the steps required to optimize sample preparation and cryopreservation with examples of the protocol with cancer and phytoplankton cells. This method provides a universal standard of sample cryo-preparation.

使用高分辨率X射线吸收光谱在低温下制备生物样品以进行形态分析

The study of elements with X-ray absorption spectroscopy (XAS) is of particular interest when studying the role of metals in biological systems. Sample ...