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>

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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。
为了成功地应用核探针分析, 必须尊重以下几点以避免潜在的陷阱。首先, 为了保持细胞的超微结构和生物化学的完整性, 必须使用低温的固定方法。其次, 还必须对厚度低于20µm 的薄试样进行分析。如果需要, 可以对 cryofixed 样品进行切片。样品应绝对保持冷冻。第三, 同样重要的是要记住, 核探针分析揭示了生物样品的二维表征。因此, 所得到的化学元素分布是一个二维投影, 可能会引入误解。这一限制将在未来绕过使用层析采集。
核探针分析也有局限性。必须强调的是, 它的主要约束是需要在真空下进行分析。因此, 必须使用细胞固定的协议, 保存细胞超微结构和生物化学的完整性。因此, 有必要测试生物样品的耐低温过程 (不加化学树脂)。这可能会限制核探针分析的使用。
另一个限制是对进行核探针分析的设施的可及性。因此, 分配的光束时间有时是有限的, 这是对这种耗时的实验的附加约束。一次收购通常需要几个小时。
该技术有别于其他分析方法, 包括显微镜、质谱 (ms)、电感耦合等离子体质谱 (ms)、液相层析 (ms) 和放射性同位素。后者确实用于估计化学元素的细胞剂量, 但它们只能提供宏观尺度的信息,即在样本的宏观量上。他们中没有一个人能像核探针分析那样, 对特定化学元素 (离子、金属或金属氧化物) 的亚细胞剂量进行精确检测和测绘。这些数据有助于进一步系统地研究剂量反应评价。
从定量的 NP 毒理学和药理学的观点来看, 对细胞内源性研究中的剂量的精确估计是至关重要的。正如所观察到的 NP 内容的大的差异所建议的那样, 在最小和最大观测浓度 (图 3) 之间有10倍的差异, 因此, 平均细胞浓度可能不是一个相关的参数来描述粒子曝光现象。这是特别真实的, 当一个阈值效应应该发生, 因为不均匀剂量暴露可能导致矛盾的观察。由于纳米粒子的分馏性质在细胞水平上出现得很清楚, 因此这项研究再次提出了基于分析全局变量的方法的相关性问题, 以解决围绕细胞的行为所暴露的问题不均匀剂量的污染物。
如这里所研究的案例所示, 在单个细胞内观察和量化 nps, 使我们能够更好地理解内生/外源元素如金属氧化物 nps 的生物蓄积。这是在生物医学领域进一步应用核动力源的一项关键任务, 因为对细胞中的 NP 分布的理解不当会导致错误的结果。
本议定书强调了使用核探针分析和其他技术的适用性, 以便将来评估与生物标本的 NP 相互作用。定量方法提供了有关此类 NPs 在检测、识别、定位和量化方面对单个单元的影响的信息。

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><tbody><tr><td>&lt;strong&gt;Cell culture&lt;/strong&gt;</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&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>11548876</td><td></td></tr><tr><td>&amp;nbsp;L-Glutamine 200 mM, 100 mL&amp;nbsp;</td><td>Invitrogen</td><td>25030024</td><td></td></tr><tr><td>Geneticin,&amp;nbsp; 20 mL</td><td>ThermoFisher Scientific</td><td>10092772</td><td></td></tr><tr><td>Trypsin-EDTA 0.25% (v/v)&amp;nbsp; 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>&lt;strong&gt;NPs preparation&lt;/strong&gt;</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>&lt;strong&gt;Sample preparation&lt;/strong&gt;</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&amp;nbsp;</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 &amp;micro;g per mL of chloroform</td></tr><tr><td>NaOH</td><td>SIGMA-ALDRICH</td><td>S5881-500G</td><td></td></tr><tr><td>&lt;strong&gt;Sample fixation&lt;/strong&gt;</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>&lt;strong&gt;Sample cryofixation&lt;/strong&gt;</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 &gt;=99%</td><td>SIGMA-ALDRICH</td><td>&amp;nbsp;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>&lt;strong&gt;Equipment&lt;/strong&gt;</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&amp;nbsp;</td><td>PD25-12-100AM</td><td></td></tr><tr><td>High-resolution Si (Li) solid-state detector,145-eVenergy resolution, @Mn-K&amp;alpha;</td><td>Oxford Instruments</td><td></td><td></td></tr><tr><td>Everhart-Thornley type secondary electron detector (SED)&amp;nbsp;</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>&lt;strong&gt;Software&lt;/strong&gt;</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&amp;uuml;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, 在无菌培养条件下数天, 然后用于不同的实验设置, 如 NPs 暴露。
化学表面修饰与荧光 (TRITC) 允许检测的2 , 以及他们的原位和体外的生活或在甲醛固定细胞的定位使用荧光显微镜。细胞核和线粒体染色使用的生命染料赫斯特33342 (蓝色的核) 和转染基质-roGFP (绿色的线粒体), 分别。这种多重染色允许 TRITC 的2 NPs 的细胞内定位 (其红色发光荧光) 20 小时后曝光。NPs 只在细胞核内没有发现的暴露细胞的细胞质中找到。NPs 被随机地定位在细胞质的周区, 完全被排除在线粒体之外 (TRITC 和 GFP 信号之间没有重叠)。
虽然荧光显微镜是非常有用的本地化 nps 内暴露的细胞, 它不能评估的确切数量的 nps 每个细胞。荧光显微镜中有关 NPs 定量化的主要难点是与 (i) 给定 NP 的荧光的数目和所选荧光在观察期间的漂白稳定性有关 (ii)单元内的 NPs 的聚合状态。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55041/55041fig1.jpg"> 图 1:体外和原位U20S 表达基质 RoGFP 的转基因细胞的荧光成像及其 TRITC2NPs标有赫斯特33342 (蓝色) 的 U2OS 单元格 (顶部), 矩阵-roGFP (绿色) 被暴露为4µg. cm-2 TRITC 标记的 "有2纳米颗粒 (红色) 为 20 h. 观察表明, 在周地区。缩放栏:10 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55041/55041fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
为了克服这些缺点, 核探针分析 techniquesprovide 了对传统光学显微镜的一种互补的方法, 因为它们对 NPs 的敏感性, 防止了从接枝中使用任何中间信号, 如荧光。染料.此外, 它们还完全是定量的, 提供有关 (i) 亚细胞生物化学含量的其他未知的数据, 以及 (ii) 单个细胞水平的 NPs 的细胞内数量。
活成像后细胞 Cryofixation。
进行核探针分析的最高限制是在真空条件下工作。我们已经制定了一个细胞固定的协议, 能够保存生物超微结构和生物标本的生物化学完整性。化学固定是已知的改变细胞的微量元素组成, 因为它需要更换他们的细胞介质的聚合物用于保存细胞的超微结构。此外, 水的去除也释放了游离离子和其他物种, 从而改变了整个样品的组成。因此, 必须优先考虑一种物理固定方法, 特别是低温方法。这些低温过程导致细胞活动的迅速停止和这, 在毫秒的时间刻度。
核探针分析显微镜和定量的 NPs 在细胞规模。
扫描透射离子显微镜 (µ控) 和粒子诱导的 X 射线发射 (µ-PIXE) 对样品进行了 cryofixation 和脱水后, 获得精确的定量数据的元素化学成分。
µ-控图像揭示了局部密度的差异, 并允许检测细胞核和细胞质等细胞结构。虽然光束横向分辨率使观察致密结构的窄至 300 nm 宽, 如薄的聚集体可见在这里的细胞质, 控方法不能区分 NP 聚合体和其他致密细胞结构。这是因为, 像透射电子显微镜 (TEM), 物理过程中, 导致传输能量的变化是传入离子与原子电子云的相互作用。不同于 TEM 分析, 但由于整个细胞体积的分析, 局部厚度变化, 防止区分高 Z 结构和局部增加细胞密度。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55041/55041fig2.jpg"> 图 2: µ-控和µ-PIXE 在低温固定 U2OS 细胞上获得的密度和元素分布的图像.U2OS 控制细胞 (上升) 与暴露的低温固定 U2OS 细胞 (暴露于4µg. cm-2 ,2 NPs, 向下) 和观察使用核探针分析/显微镜。控显微镜 (左, 灰度地图) 显示致密的内部或额外的细胞结构 (核, 盐聚合, 纳米粒子)。空间分辨率 (300 nm) 可与荧光显微镜相媲美, 并显示了周地区的 NP 聚合体等结构。然而, 仅仅基于其密度的结构的识别可能是模棱两可的。µ-PIXE 元素图的 K, P 和 Ti (热色尺度) 是互补的µ-控地图。它们可用于确保在单元格中存在 NPs。每个单元格都可以根据元素浓度进行单独分析 (请参见图 3)。比例栏:10 µm. 颜色刻度范围从最小 (蓝色) 到最大强度 (灰色) 不等。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55041/55041fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
如图 2所示, µ-控渲染允许对种群中的单个细胞和胞内子室 (如核仁和细胞核) 进行识别。不幸的是, 如前所述, 使用µ-控重建时, 无法始终检测到 NPs。
粒子诱导的 X 射线发射 (µ-PIXE) 分析不仅提供了样品的化学成分, 而且还为它们的元素映射 (图 2)。在与激振光束的相互作用过程中, 化学元素经历了原子激发----激发过程, 最终导致光子的发射, 从而呈现出激发元件的原子数的特征能量。一个特征峰谱是建立在所有发射光子事件的总和, 并被认为是一个化学签名的样品。
用于µ-PIXE 实验的标准实验装置和检测器允许同时量化所有重于 Na 的元素, 1 到10µg. g− 1干质量检测限制。测量元素浓度的准确度通常限制在20% 左右, 因为电荷收集和检测效率。
在本研究中, 通过核探针分析, 观察了钾、磷等元素的分布情况, 并用钛测图对其胞内含量进行了定量分析.化学元素映射是在光子根据记录时间的波束位置和围绕特定元素的能量窗口的选择来计算的。地图是代表的数量检测事件的光束位置, 是定量的。对噪声和背景进行了数值模拟和滤波。此外, 化学映射可用于提取定量所需的局部 PIXE 谱。
如图 2所示, 磷是 homogenously 分布在细胞中的, 与预期的一样, 在核领域的浓度要高得多。钾在细胞体积中均匀分布。钛位于细胞质周区域以团聚体的形式, 如以前观察使用荧光显微镜 (图 1)。无论其表面状态如何, NPs 都显示了相同的周本地化: 功能化 (图 1) 或本机 (图 2)。同时, 在控制细胞内没有发现钛的踪迹, 证实µ-PIXE 所观察到的钛的分布必须归因于2核粒子, 与我们先前的荧光显微镜分析一致。
此外, 如图 2所示, 可以从特定的感兴趣区域提取 nps 的细胞内分布和定量的 nps 浓度。基于控映射, 并与磷/钾分布相关, 单细胞分析是可能的。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55041/55041fig3.jpg"> 图 3: 使用µ PIXE 进行单细胞定量分析.为了确定细胞水平的元素浓度, 可以对图 2中所示的单元计算出单独的 X 射线谱。这一特性对于 NP 分析尤其有趣, 那里的细胞浓度通常在同一种群内表现出很强的变化。例如, 对于相同的平均曝光, Ti 的浓度范围从0.2 到1.8 µg. cm-2。控件对应于未经处理的细胞数量。曝光剂量: 4 µg 厘米-2。Boxplots 代表单个细胞浓度的分布, 中间值 (水平线) 和延伸到最低和最高测量值的条形。控制单元: N=14;暴露的细胞: N=16。
因此, 我们不仅对细胞中钛的平均含量进行了量化, 而且在一个特定的实验条件下, 还显示了每一个细胞中的钛分布。在这里测量的钛的中位数是相当低的 (500 ng cm-2), 与4µg. cm-2暴露剂量的细胞数量 (图 3) 和细胞间的变化很大 (Ti 浓度范围从0.2 到1.8 µg. cm-2根据分析的单元格)。我们还注意到在暴露的样品中, 如钾和钙等细胞内离子的增加, 这表明由于存在2 NPs 而引起的细胞改变稳态, 如前面的几个作者所描述的。21,27

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

Nanyang Technological University

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

Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate using the Atomic Force Microscope (AFM), can provide key insights into physical interactions present within colloidal systems. Colloidal systems are widely existent in several applications including, pharmaceuticals, foods, paints, paper, soil and minerals, detergents, printing and much more.1-3 Furthermore, colloids can exist in many states such as emulsions, foams and suspensions. Using colloidal probe nanoscopy one can obtain key information on the adhesive properties, binding energies and even gain insight into the physical stability and coagulation kinetics of the colloids present within. Additionally, colloidal probe nanoscopy can be used with biological cells to aid in drug discovery and formulation development. In this paper we describe a method for conducting colloidal probe nanoscopy, discuss key factors that are important to consider during the measurement, and show that both quantitative and qualitative data that can be obtained from such measurements.

Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems.

粒子粒子相互作用采用胶体探针纳米显微定量和定性检查

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate ...

科研

化学

Laser-induced Breakdown Spectroscopy: A New Approach for Nanoparticle's Mapping and Quantification in Organ Tissue

Emission spectroscopy of laser-induced plasma was applied to elemental analysis of biological samples. Laser-induced breakdown spectroscopy (LIBS) performed on thin sections of rodent tissues: kidneys and tumor, allows the detection of inorganic elements such as (i) Na, Ca, Cu, Mg, P, and Fe, naturally present in the body and (ii) Si and Gd, detected after the injection of gadolinium-based nanoparticles. The animals were euthanized 1&#160;to 24 hr&#160;after intravenous injection of particles. A two-dimensional scan of the sample, performed using a motorized micrometric 3D-stage, allowed the infrared laser beam exploring the surface with a lateral resolution less than 100 &#956;m. Quantitative chemical images of Gd element inside the organ were obtained with sub-mM sensitivity. LIBS offers a simple and robust method to study the distribution of inorganic materials without any specific labeling. Moreover, the compatibility of the setup with standard optical microscopy emphasizes its potential to provide multiple images of the same biological tissue with different types of response:&#160;elemental, molecular, or cellular.

Laser-induced breakdown spectroscopy performed on thin organ and tumor tissue successfully detected natural elements and artificially injected gadolinium (Gd), issued from Gd-based nanoparticles. Images of chemical elements reached a resolution of 100 &#956;m and quantitative sub-mM sensitivity. The compatibility of the setup with standard optical microscopy emphasizes its potential to provide multiple images of a same biological tissue.

激光诱导击穿光谱:对于纳米粒子的映射和定量的器官组织的新方法

Emission spectroscopy of laser-induced plasma was applied to elemental analysis of biological samples. Laser-induced breakdown spectroscopy (LIBS) ...

工程学

Detection and Recovery of Palladium, Gold and Cobalt Metals from the Urban Mine Using Novel Sensors/Adsorbents Designated with Nanoscale Wagon-wheel-shaped Pores

Developing low-cost, efficient processes for recovering and recycling palladium, gold and cobalt metals from urban mine remains a significant challenge in industrialized countries. Here, the development of optical mesosensors/adsorbents (MSAs) for efficient recognition and selective recovery of Pd(II), Au(III), and Co(II) from urban mine was achieved. A simple, general method for preparing MSAs based on using high-order mesoporous monolithic scaffolds was described. Hierarchical cubic Ia3d wagon-wheel-shaped MSAs were fabricated by anchoring chelating agents (colorants) into three-dimensional pores and micrometric particle surfaces of the mesoporous monolithic scaffolds. Findings show, for the first time, evidence of controlled optical recognition of Pd(II), Au(III), and Co(II) ions and a highly selective system for recovery of Pd(II) ions (up to ~95%) in ores and industrial wastes. Furthermore, the controlled assessment processes described herein involve evaluation of intrinsic properties (e.g., visual signal change, long-term stability, adsorption efficiency, extraordinary sensitivity, selectivity, and reusability); thus, expensive, sophisticated instruments are not required. Results show evidence that MSAs will attract worldwide attention as a promising technological means of recovering and recycling palladium, gold and cobalt metals.

Because of the importance and extensive use of palladium, gold and cobalt metals in high-technology equipment, their recovery and recycling constitute an important industrial challenge. The metal recovery system described herein is a simple, low-cost means for the effective detection, removal, and recovery of these metals from the urban mine.

检测和钯的回收，从城市矿山黄金和钴金属使用标有纳米货车车轮形的孔隙新型传感器/吸附剂

Developing low-cost, efficient processes for recovering and recycling palladium, gold and cobalt metals from urban mine remains a significant challenge in ...

Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic spread has already occurred. However, ovarian cancer has a unique pattern of metastatic spread, in that tumor implants are initially contained within the peritoneal cavity. This feature could enable, in principle, the complete resection of tumor implants with curative intent. Many of these metastatic lesions are microscopic, making them hard to identify and treat. Neutralizing such micrometastases is believed to be a major goal towards eliminating tumor recurrence and achieving long-term survival. Raman imaging with surface enhanced resonance Raman scattering nanoprobes can be used to delineate microscopic tumors with high sensitivity, due to their bright and bioorthogonal spectral signatures. Here, we describe the synthesis of two &#39;flavors&#39; of such nanoprobes: an antibody-functionalized one that targets the folate receptor &#8212; overexpressed in many ovarian cancers &#8212; and a non-targeted control nanoprobe, with&#160;distinct spectra. The nanoprobes are co-administered intraperitoneally to mouse models of metastatic human ovarian adenocarcinoma. All animal studies were approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center. The peritoneal cavity of the animals is surgically exposed, washed, and scanned with a Raman microphotospectrometer. Subsequently, the Raman signatures of the two nanoprobes are decoupled using a Classical Least Squares fitting algorithm, and their respective scores divided to provide a ratiometric signal of folate-targeted over untargeted probes. In this way, microscopic metastases are visualized with high specificity. The main benefit of this approach is that the local application into the peritoneal cavity &#8212; which can be done conveniently during the surgical procedure &#8212; can tag tumors without subjecting the patient to systemic nanoparticle exposure. False positive signals stemming from non-specific binding of the nanoprobes onto visceral surfaces can be eliminated by following a ratiometric approach where targeted and non-targeted nanoprobes with distinct Raman signatures are applied as a mixture. The procedure is currently still limited by the lack of a commercial wide-field Raman imaging camera system, which once available will allow for the application of this technique in the operating theater.

Ovarian cancer forms metastases throughout the peritoneal cavity. Here, we present a protocol to make and use folate-receptor targeted surface-enhanced resonance Raman scattering nanoprobes that reveal these lesions with high specificity via ratiometric imaging. The nanoprobes are administered intraperitoneally to living mice, and the derived images correlate well with histology.

表面增强共振拉曼散射纳米探针比比法检测微卵巢癌的意义上

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic ...

癌症研究

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity and convenient colorimetric readout. However, high densities of nanoparticles are typically needed for detection. The available synthesis-based enhancement protocols are either limited to gold and silver nanoparticles or rely on precise enzymatic control and optimization. Here, we present a protocol to enhance the colorimetric readout of gold, silver, silica, and iron oxide nanoprobes. It was observed that the colorimetric signal can be improved by up to a 10000-fold factor. The basis for such signal enhancement strategies is the chemical reduction of Au3+ to Au0. There are several chemical reactions that enable the reduction of Au3+ to Au0. In the protocol, Good&#39;s buffers and H2O2 are used and it is possible to favor the deposition of Au0 onto the surface of existing nanoprobes, in detriment of the formation of new gold nanoparticles. The protocol consists of the incubation of the microarray with a solution consisting of chloroauric acid and H2O2 in 2-(N-morpholino)ethanesulfonic acid pH 6 buffer following the nanoprobe-based detection assay. The enhancement solution can be applied to paper and glass-based sensors. Moreover, it can be used in commercially available immunoassays as demonstrated by the application of the method to a commercial allergen microarray. The signal development requires less than 5 min of incubation with the enhancement solution and the readout can be assessed by naked eye or low-end image acquisition devices such as a table-top scanner or a digital camera.

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

In this work, a protocol for signal enhancement of nanoprobe-based biosensing is presented. The protocol is based on the reduction of chloroauric acid onto the surface of existing nanoprobes that consist of gold, silver, silica or iron-oxide nanoparticles.

原位金纳米粒子合成快速 Nanoprobe 信号增强

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity ...

Processing of Bulk Nanocrystalline Metals at the US Army Research Laboratory

Given their potential for significant property improvements relative to their large grained counterparts, much work has been devoted to the continued development of nanocrystalline metals. Despite these efforts, the transition of these materials from the lab bench to actual applications has been blocked by the inability to produce large scale parts that retain the desired nanocrystalline microstructures. Following the development of a method proven to stabilize the nanosized grain structure to temperatures approaching that of the melting point for the given metal, the US Army Research Laboratory (ARL) has progressed to the next stage in the development of these materials - namely the production of large scale parts suitable for testing and evaluation in a range of relevant test environments. This report provides a broad overview of the ongoing efforts in the processing, characterization, and consolidation of these materials at ARL. In particular, focus is placed on the methodology used for producing the nanocrystalline metal powders, in both small and large-scale amounts, that are at the center of ongoing research efforts.

This paper provides a brief overview of the ongoing efforts at the Army Research Laboratory on the processing of bulk nanocrystalline metals with an emphasis on the methodologies used for the production of the novel metal powders.

美国陆军研究实验室散装纳米晶金属的加工

Given their potential for significant property improvements relative to their large grained counterparts, much work has been devoted to the continued ...

Cell Culture on Silicon Nitride Membranes and Cryopreparation for Synchrotron X-ray Fluorescence Nano-analysis

Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions and their disturbed homeostasis is involved in various diseases. State-of-the-art synchrotron X-ray fluorescence nanoprobes provide the required sensitivity and spatial resolution to elucidate the two-dimensional (2D) and three-dimensional (3D) distribution and concentration of metals inside entire cells at the organelle level. This opens new exciting scientific fields of investigation on the role of metals in the physiopathology of the cell. The cellular preparation is a key and often complex procedure, particularly for basic analysis. Although X-ray fluorescence techniques are now widespread and various preparation methods have been used, very few studies have investigated the preservation of the elemental content of cells at best, and no stepwise detailed protocol for the cryopreparation of adherent cells for X-ray fluorescence nanoprobes has been released so far. This is a description of a protocol that provides the stepwise cellular preparation for fast cryofixation to enable synchrotron X-ray fluorescence nano-analysis of cells in a frozen hydrated state when a cryogenic environment and transfer is available. In case nano-analysis has to be performed at room temperature, an additional procedure for freeze-drying the cryofixed adherent cellular preparation is provided. The proposed protocols have been successfully used in previous works, most recently in studying the 2D and 3D intracellular distribution of an organometallic compound in breast cancer cells.

Presented here is a protocol for cell culture on silicon nitride membranes and plunge-freezing prior to X-ray fluorescence imaging with a synchrotron cryogenic X-ray nanoprobe. When only room temperature nano-analysis is provided, the frozen samples can be further freeze-dried. These are critical steps to obtain information on the intracellular elemental composition.

硅氮化物膜的细胞培养和同步加速器X射线荧光纳米分析的冷冻制备

Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions ...

生物化学

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 ...

A Continuous-flow Photocatalytic Reactor for the Precisely Controlled Deposition of Metallic Nanoparticles

In this work, a novel photocatalytic reactor for the pulsed and controlled excitation of the photocatalyst and the precise deposition of metallic nanoparticles is developed. Guidelines for the replication of the reactor and its operation are provided in detail. Three different composite systems (Pt/graphene, Pt/TiO2, and Au/TiO2) with monodisperse and uniformly distributed particles are produced by this reactor, and the photodeposition mechanism, as well as the synthesis optimization strategy, are discussed. The synthesis methods and their technical aspects are described comprehensively. The role of the ultraviolet (UV) dose (in each excitation pulse) on the photodeposition process is investigated and the optimum values for each composite system are provided.

For a continuous and scalable synthesis of noble-metal-based nanocomposites, a novel photocatalytic reactor is developed and its structure, operation principles, and product quality optimization strategies are described.

一种用于金属纳米粒子精确控制沉积的连续流动光催化反应器

In this work, a novel photocatalytic reactor for the pulsed and controlled excitation of the photocatalyst and the precise deposition of metallic ...

Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the identification, localization, and characterization of nanoscale materials in biological samples is on the rise. Currently available methods, such as electron microscopy, tend to be resource-intensive, making their use prohibitive for much of the research community. Enhanced darkfield microscopy complemented with a hyperspectral imaging system may provide a solution to this bottleneck by enabling rapid and less expensive characterization of nanoparticles in histological samples. This method allows for high-contrast nanoscale imaging as well as nanomaterial identification. For this technique, histological tissue samples are prepared as they would be for light-based microscopy. First, positive control samples are analyzed to generate the reference spectra that will enable the detection of a material of interest in the sample. Negative controls without the material of interest are also analyzed in order to improve specificity (reduce false positives). Samples can then be imaged and analyzed using methods and software for hyperspectral microscopy or matched against these reference spectra in order to provide maps of the location of materials of interest in a sample. The technique is particularly well-suited for materials with highly unique reflectance spectra, such as noble metals, but is also applicable to other materials, such as semi-metallic oxides. This technique provides information that is difficult to acquire from histological samples without the use of electron microscopy techniques, which may provide higher sensitivity and resolution, but are vastly more resource-intensive and time-consuming than light microscopy.

Enhanced darkfield microscopy and hyperspectral imaging with spectral mapping enable screening, localization, and identification of nanoscale materials in histological samples with improved speed and accuracy over traditional methods. The goal of this paper is to provide methods for darkfield imaging and hyperspectral mapping of metal oxide nanoparticles in histological samples.

金属氧化物纳米颗粒的组织学样品鉴定增强暗场显微镜和高光​​谱遥感制图

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the ...

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

摘要

探索更多视频

此视频中的章节

粒子粒子相互作用采用胶体探针纳米显微定量和定性检查

激光诱导击穿光谱:对于纳米粒子的映射和定量的器官组织的新方法

检测和钯的回收，从城市矿山黄金和钴金属使用标有纳米货车车轮形的孔隙新型传感器/吸附剂

表面增强共振拉曼散射纳米探针比比法检测微卵巢癌的意义上

原位金纳米粒子合成快速 Nanoprobe 信号增强

美国陆军研究实验室散装纳米晶金属的加工

硅氮化物膜的细胞培养和同步加速器X射线荧光纳米分析的冷冻制备

单金属纳米粒子的高分辨率物理特性

一种用于金属纳米粒子精确控制沉积的连续流动光催化反应器

金属氧化物纳米颗粒的组织学样品鉴定增强暗场显微镜和高光谱遥感制图

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

摘要

探索更多视频

此视频中的章节

粒子粒子相互作用采用胶体探针纳米显微定量和定性检查

激光诱导击穿光谱:对于纳米粒子的映射和定量的器官组织的新方法

检测和钯的回收，从城市矿山黄金和钴金属使用标有纳米货车车轮形的孔隙新型传感器/吸附剂

表面增强共振拉曼散射纳米探针比比法检测微卵巢癌的意义上

原位金纳米粒子合成快速 Nanoprobe 信号增强

美国陆军研究实验室散装纳米晶金属的加工

硅氮化物膜的细胞培养和同步加速器X射线荧光纳米分析的冷冻制备

单金属纳米粒子的高分辨率物理特性

一种用于金属纳米粒子精确控制沉积的连续流动光催化反应器

金属氧化物纳米颗粒的组织学样品鉴定增强暗场显微镜和高光​​谱遥感制图

金属氧化物纳米颗粒的组织学样品鉴定增强暗场显微镜和高光谱遥感制图