Name: in situ detection and single cell quantification of metal oxide nanoparticles using nuclear microprobe analysis
Uploaded: 2018-02-03T13:00:00
Description: Watch this Scientific Journal Video about In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis at JoVE.com

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>...

<ol>
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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. ...

Research

JoVE Journal

Chemistry

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

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

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

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

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

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

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

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

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

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

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

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

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 ...

Solvothermal Synthesis of MIL-96 and UiO-66-NH2 on Atomic Layer Deposited Metal Oxide Coatings on Fiber Mats

Solvothermal Synthesis of MIL-96 and UiO-66-NH2 on Atomic Layer Deposited Metal Oxide Coatings on Fiber Mats

溶剂热合成 MIL-96 和 UiO-66-NH2在纤维垫上原子层沉积金属氧化物涂层上的合成

Metal-organic frameworks (MOFs), which contain reactive metal clusters and organic ligands allowing for large porosities and surface areas, have proven ...

High Resolution Physical Characterization of Single Metallic Nanoparticles

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 ...