我们感谢 Borderes 导演和编辑的视频。法国国家研究机构支持研究计划 TITANIUMS (全国抵抗组织 CES 2010, 编号 CESA 009 01)。CNRS 和欧洲共同体作为一项一体化活动, 在欧共体合同编号227012下提供了 "利用离子束技术 (精神) 支持公共和工业研究"。这项工作得到了玛丽居里行动的支持-初始训练网络 (蚊帐) 作为 "整合活动, 支持研究生研究与实习在工业和培训卓越" (雪碧, D1.3) 根据 EC 合同317169。C "纳米大南西部和区域住宿支持研究计划毒物-nano (n°20111201003) 和研究计划 POPRA (编号 14006636-034)。

1. 样品夹准备
<ol><li>
样品架设计和准备
	<ol>
<li>通过在 1 mm 厚的 PEEK 框架中钻取一个 5 mm x 5 mm 正方形来制造样品夹。</li>
		<li>用乙醇 70% (v/v) 冲洗清洁, 并保持无菌的盘子直到准备使用。 注意.需要一个适合细胞培养和细胞处理的样本持有者。它需要设计为细胞培养,体外观察与光学显微镜, 和核探针分析和成像。此持有者由 PEEK 帧13组成。</li>
	</ol>
</li>
	<li>样品持有人准备<ol>
<li>在100毫升氯仿中溶解1毫克的 Formvar 粉, 制备 Formvar 溶液。</li>
		<li>在金属框架上拉伸聚碳酸酯箔, 使其不呈现褶皱。</li>
		<li>使用无菌的尖端在样品夹孔周围传播 Formvar 溶液。</li>
		<li>微妙地放下样品持有人与胶水到拉伸聚碳酸酯箔 (一个5毫米 x 5 毫米正方形)。</li>
		<li>重复前面的序列, 为每个复制准备一个样本持有者。 注意.聚碳酸酯 (PC) 薄膜具有生物相容性, 厚度足够, 抗不同的协议和分析约束。 警告-氯仿是有毒的。避免吸入、吞食或接触皮肤。始终使用适当的化学通风罩和合适的过滤器。</li>
	</ol>
</li>
	<li>样品夹持器灭菌<ol>
<li>使用孵化器/搅拌器, 将安装的样品夹放在50毫升乙醇 70% (v/v) 的圆锥烧瓶中, 搅拌时在 180 rpm 和 +37 ° c 下过夜。</li>
		<li>在无菌蒸馏水中多次冲洗持液器。</li>
		<li>空气在无菌条件下晾干, 并使它们暴露在紫外线下 (生物安全工作台的杀菌灯, 第二类), 每边30分钟。</li>
		<li>保持样品在无菌12井板 (一个样品持有人每井), 直到准备使用。 注意:多个样品持有人可以储存在室温下无菌和干燥 12-井板密封膜了几天。</li>
	</ol>
</li>
</ol>2. 适当的样品持有者的细胞生长。
警告:必须在生物安全的层流工作台 (二级) 中实施议定书, 以排除污染的微生物。用手套处理抗生素 (如青霉素、链霉素)。在处理生物材料时尊重最佳做法 (细胞系, 基因修饰的人类细胞)。
关键:应检查使用的单元格线, 以确保它们没有感染支原体。
<ol><li>染 U2OS 细胞系与质粒轴承基体-roGFP 17,18,19使用转染方法按照制造商建立的协议。</li>
	<li>通过携带生物传感器的质粒来确认转染, 防止质粒丢失, 在适当培养基上选择并维持细胞。</li>
	<li>通过荧光显微镜对转染细胞进行可视化, 以确保其发生, 并确认荧光的表达以及网状和管状线粒体网络的显示19。 暂停点:细胞可以在液氮中冷冻数年。</li>
	<li>通过在适当培养基中培养细胞以获得亚汇合细胞群, 使用转染细胞。在饱和水的环境中, 在37° c、5% (v/v) CO2中生长细胞。</li>
	<li>种子细胞在浓度, 如他们在80% 汇合的一天固定。通常, 每µL 500 细胞的浓度可以使用。收获细胞使用400µL 胰蛋白酶-EDTA 0.25% (v/v) 和保持3分钟在37° c。1毫升培养基停止胰蛋白酶的作用。在 230 x g 和 +4 ° c 的情况下, 用离心法对细胞进行5分钟的颗粒化, 然后除去上清, 添加所需的新鲜培养基量。 注意.该协议适用于所有细胞类型和种子细胞数量取决于细胞大小和细胞加倍时间。 警告:避免将胰蛋白酶、抗生素和血清置于反复的冻融循环中。让他们不育将库存溶液分成等分, 并在20° c 下冷冻。将等分存储到到期日期。仔细冲洗细胞颗粒, 以完全去除胰蛋白酶。残留胰蛋白酶的痕迹会延缓和降低电镀效率。</li>
	<li>计数细胞和执行稀释与新鲜的完全培养基保持在37° c 为了获得一个细胞悬浮与500个细胞每µL。 注意:细胞悬浮的集中必须适应作为细胞类型的作用, 为了板材40µL 下落。</li>
	<li>板40µL 下降到中心的聚碳酸酯箔。小心地将样品放在2小时的 +37 ° c, 5% (v/v) CO2在饱和水的大气中。 关键:一旦样品放在孵化器, 限制尽可能多的机械运动有利于快速细胞附件。根据细胞类型, 可能需要孵育长于2小时的细胞, 以获得最佳的茨效率。检查孵化器的气氛是否充分饱和, 防止水滴蒸发。</li>
	<li>在聚碳酸酯箔上使用光学显微镜检查电池是否连接良好。轻轻地加入2毫升的新鲜完整的培养基, 并保持24小时。</li>
</ol>3. 纳米微粒的制备和曝光
注: 用四甲基罗丹明异硫氰酸酯 (TRITC) 设计、合成和化学改性荧光染料修饰的2 NPs。20,21此曲面修改允许纳米粒子检测、跟踪和定位原位和cellulo中的活和固定细胞或多细胞生物体。12,13,18
警告:纳米材料和纳米颗粒必须小心处理。避免吸入、吞食或接触皮肤。为了防止在空气中传播, 纳米颗粒被保存在溶液中 (超纯水)。
<ol><li>在超纯水中准备悬挂1毫克的− 2 NPs。使用超声波发生器和专用锥形微探针, 在 RT (750 W、20赫、振幅: 30%) 上使用强1分钟超声脉冲分散2 NPs。</li>
	<li>在培养基中的所需浓度下稀释2 NPs 以获得4µg 的曝光悬浮液. cm2 (最终浓度)。用适当的介质容量替换介质, 并在单元格上混合, 然后轻轻地将其均匀的分布在2 nps 上. 如果不添加所有的2 nps, 则同样准备控制单元。</li>
	<li>孵育细胞群为24小时在37° c, 5% (v/v) CO2和饱和水的大气。</li>
</ol>4. 甲醛固定和荧光显微镜。
<ol><li>准备一个新的溶液甲醛溶液 (粉煤灰, 4% 瓦特/v) 缓冲磷酸盐缓冲盐水 (PBS, pH 7.4)。<ol>
<li>在100毫升的 PBS 中溶解4克粉煤灰粉。将溶液加热到65° c, 同时在油烟机上使用磁铁和磁力搅拌器搅拌。增加 pH 值加1米氢氧化钠一次下降, 直到解决方案清除。将溶液冷却到室温, 并将 pH 值调整到7.4。立即使用新鲜的准备好的溶液或保持在 +4 ° c 和保护免受光。 注意:可采用预制的粉煤灰解决方案, 但即兴制备的粉煤灰保证了固定样品的固定质量和长期保存。 警告:粉煤灰是有毒的;避免吸入、吞食或接触皮肤。始终使用适当的化学通风罩和合适的过滤器。</li>
	</ol>
</li>
	<li>一旦, 潜伏期已经过去, 删除包含了2 NPs 的单元格培养基. 用新鲜培养基冲洗细胞群, 一次用 PBS (2 毫升) 清洗。去除 PBS, 用新鲜和冷的粉煤灰分快速冲洗 (4% 瓦特/五, +4 ° c)。<ol>
<li>加入粉煤灰 (2 毫升, 4% 瓦特/五, +4 ° c)。室温孵育15分钟。</li>
		<li>去除煤灰, 用 PBS 冲洗细胞 (2 毫升, 3 次, 5 分钟) 在鼓动之下。 暂停点:这些化学固定的样品可以用于 "暴跌-冻结" 程序后, 荧光成像 (转到步骤 5) 或只用于荧光成像 (转到步骤 4.3)。</li>
	</ol>
</li>
	<li>用赫斯特33342在 PBS 中孵育10分钟的细胞, 将细胞核染色。Hoescht33342用于最终浓度为 500 nM。孵化后取出溶液, 用 PBS 冲洗。 警告:赫斯特33342是单元格 permeant, 可能是有毒的。避免直接接触, 并使用手套, 同时准备和使用赫斯特33342。 关键:确保赫斯特33342着色解决方案是在无菌条件下刚准备好并保持的。</li>
	<li>使用荧光显微镜 (图 1) 处理原位和单细胞成像的固定样本。</li>
</ol>5. "骤降-冷冻" 固定和脱水
<ol><li>通过在液氮中冷却来制备铝转移板。把盘子放进一个装满液氮的盒子里, 把盘子表面保持在冰冷的氮气蒸气中。 注意:该转移板可以由任何金属板 (这里, 我们使用的铝), 可以冷却到液氮温度, 并可以进入冷冻干燥样品室。 关键:为了防止水蒸气沉积到冷板表面, 应尽可能地关闭箱体。在准备过程中储存的样品不应被液氮覆盖。</li>
	<li>在培养基中冲洗一次细胞, 然后在无菌和超纯水中再用两次, 去除残留的胞外盐。 关键:确保媒体在无菌条件下的新鲜准备和维护。在使用前在37° c 加热所有的介质。冲洗必须非常简短 (几秒钟) 和多余的液体在样品上尽可能快地去除。</li>
	<li>在三十年代的液氮冷冻 2-丁烷中, 在-150 ° c 下, 将电池浸入铝转移板上。在准备所有其他样品的过程中存储样品。当样品全部 cryofixed 时, 将转移盘置于冷冻干燥机中, 一次性转移。 关键:整个 cryofixation 过程不应超过20分钟, 以防止结构修改出现在临时存储在转移箱中的样品中。</li>
	<li>冷冻干燥样品使用以下序列: 1) 在低压和低温下执行一个主要干燥为12到 24 h (-99 ° c, 10-3毫巴), 然后 2) 执行二次干燥阶段为至少 24 h, 增加板材温度到+40 ° c, 同时保持压力低 (+ 40 ° c, 10-3毫巴)。 警告:液氮非常冷, 接触时会引起严重冻伤或眼部损伤。使用适合运输和穿戴安全设备 (低温手套, 眼睛和脸部保护) 的台式顶部容器。 警告: 2-丁烷是非常易燃的。空气的有害的污秽在这个物质的蒸发可以相当快速地到达在 +20 ° c。避免吸入、吞食或接触皮肤。使用呼吸和眼睛保护, 和防护手套。总是使用一个正常运作的化学油烟罩。可贮存于 +4 ° c。 关键:使用适当的温度计 (-150 ° c), 以监测2丁烷温度在 "暴跌冻结" 会议。2-丁烷必须保持凉爽的液体阶段。将 cryofixed 样品转移到环境大气中, 可能会因样品表面温度升高或水蒸气凝结而造成细胞损伤。因此, 转让应尽可能快, 尽可能合理 (三十年代) 暂停点:在 cryofixation 后, 样品可以在室温下存放数天, 在无菌和干燥条件下 (免受灰尘和湿气的保护)。小心地用细钳处理样品, 并把它们放在一个无菌的 12-井板密封膜。干燥剂可用于干燥大气。</li>
</ol>6. 核探针分析
注: 核探针分析是在 AIFIRA 的微线上进行的 (应用 Interdisciplinaires des Faisceaux Région 住宿) 使用互补离子束分析技术粒子诱导的 X 射线发射 (µPIXE) 和扫描透射离子显微镜 (µ-控)。该设施是基于一个 3.5 MV 粒子加速器提供的光离子束在兆伏能量范围。22,23
暂停点:AIFIRA 是一个由波尔多大学主办的离子束设施, 它提供了一个进入国家和国际小组的科学评估的建议的实验。
<ol><li>使用双面胶带将 PEEK 样品夹安装在阑板上。</li>
	<li>将板支承试样置于垂直于光束方向的竖直平面上。</li>
	<li>关闭分析室并真空分析室。</li>
	<li>扫描透射离子显微镜 (µ-控) 注: 控用于记录能量损失转化为细胞质量后的细胞区域密度图, 利用能量损失与样品的面积密度成正比的事实 (以µg. cm-2表示)。<ol>
<li>使用2兆伏特的氦 (他+) 微作为探针, 其焦距约为 300 nm, 直径和低流畅度 (2000 离子. s-1)。用平面硅探测器 (点检测器、25 mm2、11伏能量分辨率 @ 5.4 兆伏) 测量传输离子的能量, 将样品置于光束轴的后面。</li>
	</ol>
</li>
	<li>粒子诱导的 X 射线发射 (µ-PIXE) 和卢瑟福背向散射光谱 (µ-RBS)。 注: µ-Pixe 和µ-RBS 分析提供了化学成分的空间分布和量化的分微米分辨率。<ol>
<li>使用1.5 兆伏特质子 (H+) 微 (50-150 pA), 集中下来的直径1µm 和扫描在相同的细胞的利益控发现从4至8小时, 约 100 x 100 µm2。</li>
		<li>通过高分辨率硅 (Li) 固态探测器 (145 eV 能量分辨率, @Mn Kα) 从入射光束轴线的45°位置, 收集来自样品中的原子 (重于 Na) 的感应 X 射线光子发射。利用检测到的光子能量来确定发射器的性质 (如元素的 Z) 和 x 射线强度来确定元素的浓度。</li>
		<li>同时用硅探测器 (部分耗尽探测器、25 mm2、11 FWHM @ 5.4 兆伏特) 在135上收集反散射质子, 以测量传入粒子的总数, 并使 X 射线强度正常化。 关键:为了校准 X 射线探测器的响应, 使用了一组用于原子浓度量化的认证校准标准。参考材料是在6.3 µm 厚的聚酯薄膜上沉积的薄薄的原子膜的集合。发射的 X 射线能量范围从 0.6 (Li, k 线) 到20.2 凯文 (Rh, k 线)。</li>
	</ol>
</li>
</ol>7. 数据分析
<ol><li>使用ImageJ的国际律师 J 插件重构化学元素映射。24 注: 专用软件的形式为 ImageJ 的插件, 已开发加工原始核探针分析数据。原始数据对应于粒子束与细胞的相互作用所产生的事件列表, 这些单元与作为时间序列的光束位置一起记录。<ol>
<li>使用国际律师协会 J 插件排序事件根据其类型: 传输离子能量 (控), 散射离子能 (RBS) 或 X 射线光子能量 (PIXE)。然后, 分别处理每种类型的数据。</li>
		<li>计算控面积密度图</li>
		<li>计算平均 X 射线光子能量谱, 并为每一个化学元素的兴趣定义一个能量窗口, 以检索元素的空间分布。</li>
		<li>定义感兴趣的区域 (ROI) (例如单个单元格、稠密结构、聚合体、 etc) 并计算相应的 x 射线光谱。</li>
	</ol>
</li>
	<li>分析相应的 RBS 频谱, 以计算事件粒子总数25。</li>
	<li>使用 Gupix 软件26适合 X 射线频谱, 以确定每个 ROI 的元素浓度。 注意: 该方法的典型检测限制 (lod) 在1到10µg. g-1在干质量, 取决于元素的原子数 (lod 随 Z 的递减)。</li>
</ol>

<ol>
	<li>Krug, H. F., Wick, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotoxicology:+An+Interdisciplinary+Challenge.">Nanotoxicology: An Interdisciplinary Challenge.</a> Angew. Chem. Int. Ed. 50 (6), 1260-1278 (2011).</li><li>Van Hove, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+surface+science+to+nanotechnology.">From surface science to nanotechnology.</a> Catalysis Today. 113 (3-4), 133-140 (2006).</li><li>Le Trequesser, Q., Seznec, H., Delville, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functionalized+nanomaterials:+their+use+as+contrast+agents+in+bioimaging:+mono-+and+multimodal+approaches.">Functionalized nanomaterials: their use as contrast agents in bioimaging: mono- and multimodal approaches.</a> Nanotox. Rev. 2 (2), 125-169 (2013).</li><li>Oberdorster, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+assessment+for+nanotechnology+and+nanomedicine:+concepts+of+nanotoxicology.">Safety assessment for nanotechnology and nanomedicine: concepts of nanotoxicology.</a> J. Int. Med. , 89-105 (2009).</li><li>Savolainen, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotechnologies,+engineered+nanomaterials+and+occupational+health+and+safety+-+A+review.">Nanotechnologies, engineered nanomaterials and occupational health and safety - A review.</a> Saf. Sci. 48 (8), 957-963 (2010).</li><li>Savolainen, K., Alenius, H., Norppa, H., Pylkkanen, L., Tuomi, T., Kasper, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+assessment+of+engineered+nanomaterials+and+nanotechnologies+-+A+review.">Risk assessment of engineered nanomaterials and nanotechnologies - A review.</a> Toxicol. 269 (2-3), 92-104 (2010).</li><li>Arora, S., Rajwade, J. M., Paknikar, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotoxicology+and+in+vitro+studies:+The+need+of+the+hour.">Nanotoxicology and in vitro studies: The need of the hour.</a> Toxicol. . Appl. Pharm. 258 (2), 151-165 (2012).</li><li>Donaldson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+the+nanoparticles+paradox.">Resolving the nanoparticles paradox.</a> Future Med. 1 (2), 229-234 (2006).</li><li>Schaumann, G. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+fate+and+biological+effects+of+Ag-+and+TiO2-nanoparticles+in+the+environment:+The+quest+for+advanced+analytics+and+interdisciplinary+concepts.">Understanding the fate and biological effects of Ag- and TiO2-nanoparticles in the environment: The quest for advanced analytics and interdisciplinary concepts.</a> Sci Total Environ. 535, 3-19 (2014).</li><li>Olesik, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Inductively Coupled Plasma Mass Spectrometers. Treatise on Geochemistry. , (2014).</li><li>Krystek, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+on+approaches+to+bio-distribution+studies+about+gold+and+silver+engineered+nanoparticles+by+inductively+coupled+plasma+mass+spectrometry.">A review on approaches to bio-distribution studies about gold and silver engineered nanoparticles by inductively coupled plasma mass spectrometry.</a> Microchem. J. 105 (November 2011), 39-43 (2012).</li><li>Le Trequesser, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodal+correlative+microscopy+for+in+situ+detection+and+quantification+of+chemical+elements+in+biological+specimens.+Applications+to+nanotoxicology.">Multimodal correlative microscopy for in situ detection and quantification of chemical elements in biological specimens. Applications to nanotoxicology.</a> J .Chem. Biol. 8 (4), 159-167 (2015).</li><li>Le Trequesser, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+in+situ+detection+and+quantification+of+metal+oxide+nanoparticles+using+multimodal+correlative+microscopy.">Single cell in situ detection and quantification of metal oxide nanoparticles using multimodal correlative microscopy.</a> Anal. Chem. 86 (15), 7311-7319 (2014).</li><li>Ackermann, C. M., Lee, S., Chang, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+Methods+for+Imaging+Metals+in+Biology:+From+Transition+Metal+Metabolism+to+Transition+Metal+Signaling.">Analytical Methods for Imaging Metals in Biology: From Transition Metal Metabolism to Transition Metal Signaling.</a> Anal. Chem. 89 (1), 22-41 (2017).</li><li>Legin, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NanoSIMS+combined+with+fluorescence+microscopy+as+a+tool+for+subcellular+imaging+of+isotopically+labeled+platinum-based+anticancer+drugs.">NanoSIMS combined with fluorescence microscopy as a tool for subcellular imaging of isotopically labeled platinum-based anticancer drugs.</a> Chem. Sci. 5, 3135 (2014).</li><li>Lanni, E. J., Rubakhin, S. S., Sweedler, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry+imaging+and+profiling+of+single+cells.">Mass spectrometry imaging and profiling of single cells.</a> J. Proteomics. 75 (16), 5036-5051 (2012).</li><li>Waypa, G. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+triggers+subcellular+compartmental+redox+signaling+in+vascular+smooth+muscle+cells.">Hypoxia triggers subcellular compartmental redox signaling in vascular smooth muscle cells.</a> Circ. Res. 106 (3), 526-535 (2010).</li><li>Dooley, C. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Dynamic+Redox+Changes+in+Mammalian+Cells+with+Green+Fluorescent+Protein+Indicators.">Imaging Dynamic Redox Changes in Mammalian Cells with Green Fluorescent Protein Indicators.</a> J. Biol. Chem. 279 (21), 22284-22293 (2004).</li><li>Hanson, G. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+Mitochondrial+Redox+Potential+with+Redox-sensitive+Green+Fluorescent+Protein+Indicators.">Investigating Mitochondrial Redox Potential with Redox-sensitive Green Fluorescent Protein Indicators.</a> J. Biol. Chem. 279 (13), 13044-13053 (2004).</li><li>Chen, X., Mao, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Titanium+dioxide+nanomaterials:+Synthesis,+properties,+modifications+and+applications.">Titanium dioxide nanomaterials: Synthesis, properties, modifications and applications.</a> Chem. Rev. 107 (7), 2891-2959 (2007).</li><li>Simon, M., Barberet, P., Delville, M. H., Moretto, P., Seznec, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Titanium+dioxide+nanoparticles+induced+intracellular+calcium+homeostasis+modi+fi+cation+in+primary+human+keratinocytes.+Towards+an+in+vitro+explanation+of+titanium+dioxide+nanoparticles+toxicity.">Titanium dioxide nanoparticles induced intracellular calcium homeostasis modi fi cation in primary human keratinocytes. Towards an in vitro explanation of titanium dioxide nanoparticles toxicity.</a> Nanotox. 5 (June), 125-139 (2011).</li><li>Sorieul, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ion+beam+facility+for+multidisciplinary+research.">An ion beam facility for multidisciplinary research.</a> Nucl. Instr. Meth. Phys. Res., B. 332, 68-73 (2014).</li><li>Barberet, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+results+obtained+using+the+CENBG+nanobeam+line:+Performances+and+applications.">First results obtained using the CENBG nanobeam line: Performances and applications.</a> Nucl Instr Meth Phys Res B. 269 (20), 2163-2167 (2011).</li><li>Dev&#232;s, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ImageJ+plugin+for+ion+beam+imaging+and+data+processing+at+AIFIRA+facility.">An ImageJ plugin for ion beam imaging and data processing at AIFIRA facility.</a> Nucl. Instr. Meth. Phys. Res. B. 348, 62-67 (2015).</li><li>Mayer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> SIMNRA User's Guide, Report IPP 9/113. , (1997).</li><li>Campbell, J. L., Boyd, N. I., Grassi, N., Bonnick, P., Maxwell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Guelph+PIXE+software+package+IV.">The Guelph PIXE software package IV.</a> Nucl. Instr. Meth. Phys. Res. B. 268 (20), 3356-3363 (2010).</li><li>Yu, K., Chang, S., Park, S. J., Lim, J., Lee, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Titanium+Dioxide+Nanoparticles+Induce+Endoplasmic+Reticulum+Stress-Mediated+Autophagic+Cell+Death+via+Mitochondria-+Associated+Endoplasmic+Reticulum+Membrane+Disruption+in+Normal+Lung+Cells.">Titanium Dioxide Nanoparticles Induce Endoplasmic Reticulum Stress-Mediated Autophagic Cell Death via Mitochondria- Associated Endoplasmic Reticulum Membrane Disruption in Normal Lung Cells.</a> PLoS ONE. , 1-17 (2015).</li></ol>

作者没有什么可透露的。

我们描述一种提供有用信息的方法, 超出了其他成像技术的可能, 特别是在亚细胞层面。除了成像能力外, 核探针分析还提供了在生物样品的组成中加入化学元素数量的可能性。在目前的工作中, 我们研究了人类细胞的数量, 并专注于分析一个选定的感兴趣的区域的基础上的一个单一的细胞暴露于2 NPs。它与其他技术的结合提供了形态学细胞成像和精确的元素化学成分的定量数据。
<p class=...

细胞稳态是由不同的微量元素 (离子、金属、外源性无机化合物) 的吸收控制、同化和细胞内定位决定的。这些组分频繁地是以踪影的形式, 但然而可能有可观的冲击在系统生理。因此, 研究细胞生物化学在正常和病理/强调的情况是一个关键步骤, 以全面了解细胞代谢机制。因此, 发展成像和分析技术, 使调查的细胞内的化学丰度, 结构组织及其相关的代谢功能成为必要。很少有方法能够提供一个原位定量的关于某一样品的化学性质的信息。除了以散装形式分析样本的方法外,原位分析在不丢失质量和结构信息的情况下考虑生物样品的完整性, 从而保存它们的化学成分 (微量元素和离子), 并蛋白质.此外, 随着 nanosciences 的不断发展, 改进的成像和分析方法的环境监测的细胞规模将是必要的观察和量化纳米对象的行为和相互作用。1
纳米粒子 (NPs) 被定义为在1和 100 nm 范围内至少有一个面部尺寸的物体。2由于其特殊的物理化学性质, NPs 在工业中得到了广泛的应用。NPs 用于生物应用和纳米。3,4尽管 NPs 有许多物理化学特性, 但它们可能会产生一些对人类健康和环境有不利影响的风险。这些风险可以由长期和重复暴露在不同浓度水平, 这是尚未明确确定。5,6,7,8特别是在细胞内的 NPs 的命运和相关的细胞反应, 到目前为止, 尚未完全描述。这在某种程度上是由于允许检测和量化单个单元中内化 NPs 的方法的稀缺性。9
用于估计纳米粒细胞剂量的经典分析工具是 microscopies、质谱 (ms)、电感耦合等离子体 ms (ICP)10、11和液相色谱 ms (LC), 但它们只提供宏观尺度上的有用信息。没有使用分馏方法, 它们都不能精确地评估亚细胞 nps 的含量, 也不能提供 nps 的分布。对剂量反应的系统的评估因而是不可能的以这些方法, 相对于基于原子光谱学的方法例如核探针分析12,13, 同步辐射 X 射线荧光显微镜14和二次离子质谱法 (西姆斯)。15,16这些方法特别有趣, 因为它们补充了使用荧光显微镜进行的观察, 特别是当 NPs 不能被标记为荧光分子, 因此在它们的原生状态下进行研究。在某种程度上, 即使当 NPs 与荧光接枝, (i) 量化仍然是困难的, 因为每个 np 的标签水平是未知的和 (ii) 的化学修饰的 np 表面可能改变其细胞分布。
在这篇文章中, 我们着重于一种基于核探针技术结合的方法, 目的是对主要、次要和微量浓度的生物标本的形态和元素组成进行成像。
核探针分析特别适用于生物组织中微量化学元素的测定。两个光束横向分辨率 (0.3 到1µm) 和灵敏度的化学元素检测 (从1到10µg. g-1干质量) 非常适合于细胞水平的研究。核探针技术是基于粒子检测 (光子, 电子, 离子) 发射后, 离子束 (通常运行在电子伏特能量) 与原子的存在, 在样品。在细胞中发生的相互作用主要是: 1) 原子的激发/电离后, 在原子返回到其基本状态后的光子发射;和 2) 传入粒子的扩散导致其能量和方向的变化。发射粒子能量的测量 allowsthe 参与相互作用的原子的识别。要执行元素的映射, 离子微在样本表面上反复扫描, 通常在大约100的区域中, 100 µm2包含多个单元格。发射的粒子被探测到, 它们的能量被记录在每个光束位置。根据光束位置对粒子进行分类, 从而确定负责此类粒子发射的结构是数据处理的目的。在这里, 我们精确地描述了一种基于荧光显微镜和核探针分析的方法来检测以及在细胞和亚细胞尺度上量化外源 NPs, 以研究 NP 相互作用与生活的后果系统.我们将特别侧重于这种方法提供的机会, 从原位定量的角度来看, 在亚细胞水平上, 二氧化钛纳米粒子 (在2 NPs) 中聚合。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>U2OS</td>
			<td>ATCC, LGC STANDARDS</td>
			<td>ATCC HTB-96</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium MCCOY 5A w/o L-Glutamine</td>
			<td>Dominique DUTSCHER</td>
			<td>L0211-500</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS 500 mL</td>
			<td>Dominique DUTSCHER</td>
			<td>500105U</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>11548876</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;L-Glutamine 200 mM, 100 mL&#160;</td>
			<td>Invitrogen</td>
			<td>25030024</td>
			<td></td>
		</tr>
		<tr>
			<td>Geneticin,&#160; 20 mL</td>
			<td>ThermoFisher Scientific</td>
			<td>10092772</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.25% (v/v)&#160; 500 mL</td>
			<td>ThermoFisher Scientific</td>
			<td>11570626</td>
			<td></td>
		</tr>
		<tr>
			<td>Viromer Red</td>
			<td>Lipocalyx</td>
			<td>VR-01LB-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrix-roGFP Plasmid</td>
			<td>AddGene</td>
			<td>#49437</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>ThermoFisher Scientific</td>
			<td>H3570</td>
			<td>Handle with care</td>
		</tr>
		<tr>
			<td>NPs preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TiO2 P25 AEROXIDE</td>
			<td>Degussa/Evonik</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylrhodamine isothiocyanate (TRITC)</td>
			<td>SIGMA-ALDRICH</td>
			<td>T3163</td>
			<td>Surface modification of NPs</td>
		</tr>
		<tr>
			<td>Sample preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate foil</td>
			<td>Goodfellow</td>
			<td>CT301020</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyether Ether Ketone support (PEEK)</td>
			<td>Matechplast</td>
			<td>A-239-4047</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, ACS absolute</td>
			<td>SIGMA-ALDRICH</td>
			<td>02860-6x1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Chlorform, Anhydrous, 99%</td>
			<td>SIGMA-ALDRICH</td>
			<td>372978-1L&#160;</td>
			<td>Caution toxic</td>
		</tr>
		<tr>
			<td>Formvar 100 g</td>
			<td>Agar Scientific</td>
			<td>AGR1201</td>
			<td>Harmful. Use in a concentration of 10 &#181;g per mL of chloroform</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>SIGMA-ALDRICH</td>
			<td>S5881-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sample fixation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Powder, 95% Paraformaldehyde</td>
			<td>SIGMA-ALDRICH</td>
			<td>158127-500G</td>
			<td>Caution toxic. Use as a 4% solution in PBS</td>
		</tr>
		<tr>
			<td>PBS (pH 7.4, without Ca2+ and Mg2+)</td>
			<td>ThermoFisher Scientific</td>
			<td>11503387</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Gold Antifade Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>P36934</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>SIGMA-ALDRICH</td>
			<td>93443</td>
			<td>Harmful</td>
		</tr>
		<tr>
			<td>Sample cryofixation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td>air liquids sante</td>
			<td></td>
			<td>Harmful</td>
		</tr>
		<tr>
			<td>Methylbutane &#62;=99%</td>
			<td>SIGMA-ALDRICH</td>
			<td>&#160;M32631-1L</td>
			<td>Caution toxic</td>
		</tr>
		<tr>
			<td>Aluminium transfer plate</td>
			<td>Home-made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled and deionized water</td>
			<td>Home-made</td>
			<td></td>
			<td>Produced in the laboratory using the Barnstead Smart2Pure system</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>VWR</td>
			<td>52858-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Barnstead Smart2Pure</td>
			<td>ThermoFisher Scientific</td>
			<td>50129870</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety bench, class II</td>
			<td>ThermoFisher Scientific</td>
			<td>MSC-Advantage</td>
			<td></td>
		</tr>
		<tr>
			<td>TC20 automated cell counter</td>
			<td>Biorad</td>
			<td>145-0102SP</td>
			<td></td>
		</tr>
		<tr>
			<td>Counting slides 2 wells</td>
			<td>Biorad</td>
			<td>1450016</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPS detector, 25 mm2, 12 keV energy resolution @5.5 MeV</td>
			<td>Canberra&#160;</td>
			<td>PD25-12-100AM</td>
			<td></td>
		</tr>
		<tr>
			<td>High-resolution Si (Li) solid-state detector,145-eVenergy resolution, @Mn-K&#945;</td>
			<td>Oxford Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Everhart-Thornley type secondary electron detector (SED)&#160;</td>
			<td>Orsay Physics</td>
			<td>1-SED</td>
			<td></td>
		</tr>
		<tr>
			<td>XRF Calibration Standard sodium or Chlorine as NaCl</td>
			<td>Micromatter</td>
			<td>34381</td>
			<td></td>
		</tr>
		<tr>
			<td>XRF Calibration Standard Magnesium as MgF2</td>
			<td>Micromatter</td>
			<td>34382</td>
			<td></td>
		</tr>
		<tr>
			<td>XRF Calibration Standard Aluminium as Al metal</td>
			<td>Micromatter</td>
			<td>34383</td>
			<td></td>
		</tr>
		<tr>
			<td>XRF Calibration Standard Silicon as SiO</td>
			<td>Micromatter</td>
			<td>34384</td>
			<td></td>
		</tr>
		<tr>
			<td>XRF Calibration Standard Sulfur as CuSx</td>
			<td>Micromatter</td>
			<td>34385</td>
			<td></td>
		</tr>
		<tr>
			<td>XRF Calibration Standard Calcium as CaF2</td>
			<td>Micromatter</td>
			<td>34387</td>
			<td></td>
		</tr>
		<tr>
			<td>XRF Calibration Standard Titanium as Ti metal</td>
			<td>Micromatter</td>
			<td>34388</td>
			<td></td>
		</tr>
		<tr>
			<td>XRF Calibration Standard Iron as Fe metal</td>
			<td>Micromatter</td>
			<td>34389</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator 750W</td>
			<td>Sonics Materials</td>
			<td>11743619</td>
			<td></td>
		</tr>
		<tr>
			<td>3MM microprobe</td>
			<td>Bioblock scientific</td>
			<td>220-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Lyophilizer in vacuum</td>
			<td>Elexience</td>
			<td>EK3147</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical microscope Zeiss AxioObserver Z1</td>
			<td>Carl Zeiss MicroImaging, GmbH</td>
			<td>431006-9901</td>
			<td></td>
		</tr>
		<tr>
			<td>Motorized stage xy</td>
			<td>Carl Zeiss MicroImaging, GmbH</td>
			<td>432031-9902</td>
			<td></td>
		</tr>
		<tr>
			<td>EC Plan-Neofluar 20X, NA 0.50 Ph2 M27 objective</td>
			<td>Carl Zeiss MicroImaging, GmbH</td>
			<td>420351-9910</td>
			<td></td>
		</tr>
		<tr>
			<td>Plan-Apochromat 63X, NA 1,40 Ph3M27 objective</td>
			<td>Carl Zeiss MicroImaging, GmbH</td>
			<td>420781-9910</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss filterset 02</td>
			<td>Carl Zeiss MicroImaging, GmbH</td>
			<td>488002-9901</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss filterset 38HE</td>
			<td>Carl Zeiss MicroImaging, GmbH</td>
			<td>489038-9901</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss filterset 31</td>
			<td>Carl Zeiss MicroImaging, GmbH</td>
			<td>000000-1031-350</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemical fume hood</td>
			<td>Erlab</td>
			<td>Captair SD321</td>
			<td></td>
		</tr>
		<tr>
			<td>Particle accelerator</td>
			<td>HVEE</td>
			<td>singletron</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>National Institutes of health, USA</td>
			<td>ImageJ 1.51</td>
			<td></td>
		</tr>
		<tr>
			<td>SimNRA software</td>
			<td>Max-Planck-Institut f&#252;r Plasmaphysik, Germany</td>
			<td>SIMNRA 6.06</td>
			<td></td>
		</tr>
		<tr>
			<td>Gupix software</td>
			<td>Guelph university, Canada</td>
			<td>GUPIXWIN 2.2.4</td>
			<td></td>
		</tr>
	</tbody>
</table>

in situ detection and single cell quantification of metal oxide nanoparticles using nuclear microprobe analysis

基于化学元素成像的微分析技术使细胞级化学成分的定位和定量化。它们为生物系统的表征提供了新的可能性, 特别适合于检测、定位和量化金属氧化物纳米粒子在生物学标本和环境中的存在。事实上, 这些技术都符合 (i) 灵敏度的相关要求 (从1到10µg. g-1的干质量), (二) 微米范围空间分辨率, 和 (三) 多元素检测。鉴于这些特点, 微化学元件成像可以有力地补充常规成像技术, 如光学和荧光显微镜。本协议描述了如何对暴露在二氧化钛纳米粒子上的培养细胞 (U2OS) 进行核探针分析。细胞必须生长, 并直接暴露在一个专门设计的样品持有人在光学显微镜和核探针分析阶段。对样品进行深低温固定, 既保护细胞组织, 又保留化学元素分布。在样品上进行的同步核探针分析 (扫描透射离子显微镜、卢瑟福背向散射光谱和粒子诱导的 X 射线发射) 提供了有关细胞密度的信息, 本地分布化学成分, 以及纳米颗粒的细胞含量。在生物学中对这种分析工具的需求越来越大, 尤其是在理学和纳米的新环境中, 我们必须加深对纳米粒子和生物样品相互作用的理解。特别是, 由于核探针分析不要求纳米颗粒被贴上标签, 纳米粒子的丰度可以量化到细胞中单个细胞的水平, 而不依赖于它们的表面状态。

荧光标记的细胞培养和荧光成像2NPs
我们设计了一个样本持有人适应细胞培养, 细胞处理以及多模态分析。具体地说, 这是重要的是, 持有人允许常规光学显微镜以及核探针分析和成像。本样品持有人是基于 2-µm 厚聚碳酸酯箔沉积在一个 PEEK 框架。细胞直接生长在聚碳酸酯上, 在无?...

Watch this Scientific Journal Video about In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis at JoVE.com

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

我们描述的程序, 检测的化学元素存在的原位在人类细胞以及他们的体外量化。该方法非常适合于任何细胞类型, 特别适用于在体外金属氧化物纳米颗粒的照射下单细胞的化学分析。

原位金属氧化物纳米粒子的核探针检测与单细胞定量研究

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. ...

in-situ-detection-single-cell-quantification-metal-oxide

Research

JoVE Journal

Chemistry

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

7.0K 次观看.  Université de Bordeaux. 我们描述的程序, 检测的化学元素存在的原位在人类细胞以及他们的体外量化。该方法非常适合于任何细胞类型, 特别适用于在体外金属氧化物纳米颗粒的照射下单细胞的化学分析。

视频: In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

Using Polystyrene-block-poly(acrylic acid)-coated Metal Nanoparticles as Monomers for Their Homo- and Co-polymerization

We present a template-free method for &#8220;polymerizing&#8221; nanoparticles into long chains without side branches. A variety of nanoparticles are encapsulated in polystyrene-block-poly(acrylic acid) (PSPAA) shells and then used as monomers for their self-assembly. Spherical PSPAA micelles upon acid treatment are known to assemble into cylindrical micelles. Exploiting this tendency, the core-shell nanoparticles are induced to aggregate, coalesce, and then transform into long chains. When more than one type of nanoparticles are used, random and block &#8220;copolymers&#8221; of nanoparticles can be obtained. Detailed procedures are reported for the PSPAA encapsulation of nanoparticles, homo- and co-polymerization of the core-shell nanoparticles, separation and purification of the resulting nanoparticle chains. Transformations of single-line chains into double- and triple-line chains are also presented. The synergy between the polymer shell and the embedded nanoparticles leads to an unusual chain-growth polymerization mode, giving long nanoparticle chains that are distinct from the products of the traditional step-growth aggregation process.

Using Polystyrene-block-polyacrylic acid-coated Metal Nanoparticles as Monomers for Their Homo- and Co-polymerization

We report protocols for &#8220;polymerizing&#8221; various types of polymer-encapsulated metal nanoparticles into long chains of &#8220;homo-&#8220; and &#8220;co-polymers&#8221;.

使用聚苯乙烯<em&gt;块</em&gt; - 聚（丙烯酸）涂层的金属纳米颗粒作为单体他们均聚物和共聚物聚合

We present a template-free method for &#8220;polymerizing&#8221; nanoparticles into long chains without side branches. A variety of nanoparticles are ...

科研

化学

Reverse Microemulsion-mediated Synthesis of Monometallic and Bimetallic Early Transition Metal Carbide and Nitride Nanoparticles

A reverse microemulsion is used to encapsulate monometallic or bimetallic early transition metal oxide nanoparticles in microporous silica shells. The silica-encapsulated metal oxide nanoparticles are then carburized in a methane/hydrogen atmosphere at temperatures over 800 &#176;C to form silica-encapsulated early transition metal carbide nanoparticles. During the carburization process, the silica shells prevent the sintering of adjacent carbide nanoparticles while also preventing the deposition of excess surface carbon. Alternatively, the silica-encapsulated metal oxide nanoparticles can be nitridized in an ammonia atmosphere at temperatures over 800 &#176;C to form silica-encapsulated early transition metal nitride nanoparticles. By adjusting the reverse microemulsion parameters, the thickness of the silica shells, and the carburization/nitridation conditions, the transition metal carbide or nitride nanoparticles can be tuned to various sizes, compositions, and crystal phases. After carburization or nitridation, the silica shells are then removed using either a room-temperature aqueous ammonium bifluoride solution or a 0.1 to 0.5 M NaOH solution at 40-60 &#176;C. While the silica shells are dissolving, a high surface area support, such as carbon black, can be added to these solutions to obtain supported early transition metal carbide or nitride nanoparticles. If no high surface area support is added, then the nanoparticles can be stored as a nanodispersion or centrifuged to obtain a nanopowder.

A &#8220;removable ceramic coating method&#8221; is presented in visual format for the synthesis of non-sintered and metal-terminated monometallic and bimetallic early transition metal carbide and nitride nanoparticles with tunable sizes and crystal structures.

单金属和双金属前过渡金属碳化物和氮化物纳米粒子的反相微乳液介导的合成

A reverse microemulsion is used to encapsulate monometallic or bimetallic early transition metal oxide nanoparticles in microporous silica shells. The ...

Generation of Zerovalent Metal Core Nanoparticles Using n-(2-aminoethyl)-3-aminosilanetriol

In this work, a facile one-pot reaction for the formation of metal nanoparticles in a water solution through the use of n-(2-aminoethyl)-3-aminosilanetriol is presented. This compound can be used to effectively reduce and complex metal salts into metal core nanoparticles coated with the compound. By controlling the concentrations of salt and silane one is able to control reaction rates, particle size, and nanoparticle coating. The effects of these changes were characterized through transmission electron microscopy (TEM), UV-Vis spectrometry (UV-Vis), Nuclear Magnetic Resonance spectroscopy (NMR) and Fourier Transform Infrared spectroscopy (FTIR). A unique aspect to this reaction is that usually silanes hydrolyze and cross-link in water; however, in this system the silane is water-soluble and stable. It is known that silicon and amino moieties can form complexes with metal salts. The silicon is known to extend its coordination sphere to form penta- or hexa-coordinated species. Furthermore, the silanol group can undergo hydrolysis to form a Si-O-Si silica network, thereby transforming the metal nanoparticles into a functionalized nanocomposites.

Generation of Zerovalent Metal Core Nanoparticles Using n-2-aminoethyl-3-aminosilanetriol

A novel method for metal core nanoparticle synthesis using a water stable silanol is described.

使用N-零价金属芯纳米粒子的生成（2-氨基乙基）-3- aminosilanetriol

In this work, a facile one-pot reaction for the formation of metal nanoparticles in a water solution through the use of ...

Photochemical Oxidative Growth of Iridium Oxide Nanoparticles on CdSe@CdS Nanorods

We demonstrate a procedure for the photochemical oxidative growth of iridium oxide catalysts on the surface of seeded cadmium selenide-cadmium sulfide (CdSe@CdS) nanorod photocatalysts. Seeded rods are grown using a colloidal hot-injection method and then moved to an aqueous medium by ligand exchange. CdSe@CdS nanorods, an iridium precursor and other salts are mixed and illuminated. The deposition process is initiated by absorption of photons by the semiconductor particle, which results with formation of charge carriers that are used to promote redox reactions. To insure photochemical oxidative growth we used an electron scavenger. The photogenerated holes oxidize the iridium precursor, apparently in a mediated oxidative pathway. This results in the growth of high quality crystalline iridium oxide particles, ranging from 0.5 nm to about 3 nm, along the surface of the rod. Iridium oxide grown on CdSe@CdS heterostructures was studied by a variety of characterization methods, in order to evaluate its characteristics and quality. We explored means for control over particle size, crystallinity, deposition location on the CdS rod, and composition. Illumination time and excitation wavelength were found to be key parameters for such control. The influence of different growth conditions and the characterization of these heterostructures are described alongside a detailed description of their synthesis. Of significance is the fact that the addition of iridium oxide afforded the rods astounding photochemical stability under prolonged illumination in pure water (alleviating the requirement for hole scavengers).

A protocol for the photochemical oxidative growth of small crystalline iridium oxide nanoparticles on the surface of CdSe@CdS seeded rod nanoparticles is presented.

铱氧化物纳米颗粒对硒化镉硫化镉@纳米棒光化学氧化增长

We demonstrate a procedure for the photochemical oxidative growth of iridium oxide catalysts on the surface of seeded cadmium selenide-cadmium sulfide ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

使用酶法，化学（甲基化）和物理（质谱，核磁共振）技术，植物墙Heteroxylans测序

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Aerosol-assisted Chemical Vapor Deposition of Metal Oxide Structures: Zinc Oxide Rods

Whilst columnar zinc oxide (ZnO) structures in the form of rods or wires have been synthesized previously by different liquid- or vapor-phase routes, their high cost production and/or incompatibility with microfabrication technologies, due to the use of pre-deposited catalyst-seeds and/or high processing temperatures exceeding 900 &#176;C, represent a drawback for a widespread use of these methods. Here, however, we report the synthesis of ZnO rods via a non-catalyzed vapor-solid mechanism enabled by using an aerosol-assisted chemical vapor deposition (CVD) method at 400 &#176;C with zinc chloride (ZnCl2) as the precursor and ethanol as the carrier solvent. This method provides both single-step formation of ZnO rods and the possibility of their direct integration with various substrate types, including silicon, silicon-based micromachined platforms, quartz, or high heat resistant polymers. This potentially facilitates the use of this method at a large-scale, due to its compatibility with state-of-the-art microfabrication processes for device manufacture. This report also describes the properties of these structures (e.g., morphology, crystalline phase, optical band gap, chemical composition, electrical resistance) and validates its gas sensing functionality towards carbon monoxide.

Columnar zinc oxide structures in the form of rods are synthesized via aerosol-assisted chemical vapor deposition without the use of pre-deposited catalyst-seed particles. This method is scalable and compatible with various substrates based on either silicon, quartz or polymers.

金属氧化物结构的气溶胶辅助化学气相沉积: 氧化锌棒

Whilst columnar zinc oxide (ZnO) structures in the form of rods or wires have been synthesized previously by different liquid- or vapor-phase routes, ...

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog (Xenopus laevis) Embryos

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic development. To enable single-cell investigations directly in live embryos, new analytical approaches are needed, particularly those that are sensitive, selective, quantitative, robust, and scalable to different cell sizes. Here, we present a protocol that enables the in situ analysis of metabolism in single cells in freely developing embryos of the South African clawed frog (Xenopus laevis), a powerful model in cell and developmental biology. This approach uses a capillary microprobe to aspirate a defined portion from single identified cells in the embryo, leaving neighboring cells intact for subsequent analysis. The collected cell content is analyzed by a microscale capillary electrophoresis electrospray ionization (CE-ESI) interface coupled to a high-resolution tandem mass spectrometer. This approach is scalable to various cell sizes and compatible with the complex three-dimensional structure of the developing embryo. As an example, we demonstrate that microprobe single-cell CE-ESI-MS enables the elucidation of metabolic cell heterogeneity that unfolds as a progenitor cell gives rise to descendants during development of the embryo. Besides cell and developmental biology, the single-cell analysis protocols described here are amenable to other cell sizes, cell types, or animal models.

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog Xenopus laevis Embryos

We describe steps that enable fast in situ sampling of a small portion of an individual cell with high precision and minimal invasion using capillary-based micro-sampling, to facilitate chemical characterization of a snapshot of metabolic activity in live embryos using a custom-built single cell capillary electrophoresis and mass spectrometry platform.

活蛙 (爪蟾) 胚胎单细胞代谢的微探针毛细管电泳质谱

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic ...

Ligand-Mediated Nucleation and Growth of Palladium Metal Nanoparticles

The size, size distribution and stability of colloidal nanoparticles are greatly affected by the presence of capping ligands. Despite the key contribution of capping ligands during the synthesis reaction, their role in regulating the nucleation and growth rates of colloidal nanoparticles is not well understood. In this work, we demonstrate a mechanistic investigation of the role of trioctylphosphine (TOP) in Pd nanoparticles in different solvents (toluene and pyridine) using in situ SAXS and ligand-based kinetic modeling. Our results under different synthetic conditions reveal the overlap of nucleation and growth of Pd nanoparticles during the reaction, which contradicts the LaMer-type nucleation and growth model. The model accounts for the kinetics of Pd-TOP binding for both, the precursor and the particle surface, which is essential to capture the size evolution as well as the concentration of particles in situ. In addition, we illustrate the predictive power of our ligand-based model through designing the synthetic conditions to obtain nanoparticles with desired sizes. The proposed methodology can be applied to other synthesis systems and therefore serves as an effective strategy for predictive synthesis of colloidal nanoparticles.

The main goal of this work is to elucidate the role of capping agents in regulating the size of palladium nanoparticles by combining in situ small angle x-ray scattering (SAXS) and ligand-based kinetic modeling.

配体介导成核与钯金属纳米粒子的生长

The size, size distribution and stability of colloidal nanoparticles are greatly affected by the presence of capping ligands. Despite the key contribution ...

High Resolution Physical Characterization of Single Metallic Nanoparticles

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single nanometer-scale pore. The signal is characteristic of the molecule&#39;s physicochemical properties and its interactions with the pore. We demonstrate that the nanopore formed by the bacterial protein exotoxin Staphylococcus aureus alpha hemolysin (&#945;HL) can detect polyoxometalates (POMs, anionic metal oxygen clusters), at the single molecule limit. Moreover, multiple degradation products of 12-phosphotungstic acid POM (PTA, H3PW12O40) in solution are simultaneously measured. The single molecule sensitivity of the nanopore method allows for POMs to be characterized at significantly lower concentrations than required for nuclear magnetic resonance (NMR) spectroscopy. This technique could serve as a new tool for chemists to study the molecular properties of polyoxometalates or other metallic clusters, to better understand POM synthetic processes, and possibly improve their yield. Hypothetically, the location of a given atom, or the rotation of a fragment in the molecule, and the metal oxidation state could be investigated with this method. In addition, this new technique has the advantage of allowing the real-time monitoring of molecules in solution.&#160;

Here, we present a protocol to detect discrete metal oxygen clusters, polyoxometalates (POMs), at the single molecule limit using a biological nanopore-based electronic platform. The method provides a complementary approach to traditional analytical chemistry tools used in the study of these molecules.

单金属纳米粒子的高分辨率物理特性

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single ...

Molten-Salt Synthesis of Complex Metal Oxide Nanoparticles

The development of feasible synthesis methods is critical for the successful exploration of novel properties and potential applications of nanomaterials. Here, we introduce the molten-salt synthesis (MSS) method for making metal oxide nanomaterials. Advantages over other methods include its simplicity, greenness, reliability, scalability, and generalizability. Using pyrochlore lanthanum hafnium oxide (La2Hf2O7) as a representative, we describe the MSS protocol for the successful synthesis of complex metal oxide nanoparticles (NPs). Furthermore, this method has the unique ability to produce NPs with different material features by changing various synthesis parameters such as pH, temperature, duration, and post-annealing. By fine-tuning these parameters, we are able to synthesize highly uniform, non-agglomerated, and highly crystalline NPs. As a specific example, we vary the particle size of the La2Hf2O7&#160;NPs by changing the concentration of the ammonium hydroxide solution used in the MSS process, which allows us to further explore the effect of particle size on various properties. It is expected that the MSS method will become a more popular synthesis method for nanomaterials and more widely employed in the nanoscience and nanotechnology community in the upcoming years.

Here, we demonstrate a unique, relatively low-temperature, molten-salt synthesis method for preparing uniform complex metal oxide lanthanum hafnate nanoparticles.

复合金属氧化物纳米粒子的熔融盐合成

The development of feasible synthesis methods is critical for the successful exploration of novel properties and potential applications of nanomaterials. ...