方法文章

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

DOI：

10.3791/55041

⸱

2018年2月3日

 ,  ,  ,  ,  ,  ,  , 
1Centre d'Etudes Nucléaires Bordeaux Gradignan (CENBG), Université de Bordeaux, 2Centre d'Etudes Nucléaires Bordeaux Gradignan (CENBG), CNRS, 3Institut de Chimie de la Matière Condens é e de Bordeaux (ICMCB), CNRS, 4Institut de Chimie de la Matière Condens é e de Bordeaux (ICMCB), Université de Bordeaux

* These authors contributed equally

本文内容

摘要

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We describe a procedure for the detection of chemical elements present in situ in human cells as well as their in vitro quantification. The method is well-suited to any cell type and is particularly useful for quantitative chemical analyses in single cells following in vitro metal oxide nanoparticles exposure.

摘要

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Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. They offer new possibilities for the characterization of living systems and are particularly appropriate for detecting, localizing and quantifying the presence of metal oxide nanoparticles both in biological specimens and the environment. Indeed, these techniques all meet relevant requirements in terms of (i) sensitivity (from 1 up to 10 µg.g-1 of dry mass), (ii) micrometer range spatial resolution, and (iii) multi-element detection. Given these characteristics, microbeam chemical element imaging can powerfully complement routine imaging techniques such as optical and fluorescence microscopy. This protocol describes how to perform a nuclear microprobe analysis on cultured cells (U2OS) exposed to titanium dioxide nanoparticles. Cells must grow on and be exposed directly in a specially designed sample holder used on the optical microscope and in the nuclear microprobe analysis stages. Plunge-freeze cryogenic fixation of the samples preserves both the cellular organization and the chemical element distribution. Simultaneous nuclear microprobe analysis (scanning transmission ion microscopy, Rutherford backscattering spectrometry and particle induced X-ray emission) performed on the sample provides information about the cellular density, the local distribution of the chemical elements, as well as the cellular content of nanoparticles. There is a growing need for such analytical tools within biology, especially in the emerging context of Nanotoxicology and Nanomedicine for which our comprehension of the interactions between nanoparticles and biological samples must be deepened. In particular, as nuclear microprobe analysis does not require nanoparticles to be labelled, nanoparticle abundances are quantifiable down to the individual cell level in a cell population, independently of their surface state.

引言

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Cellular homeostasis is determined by the uptake control, assimilation, and intracellular localization of different trace elements (ions, metals, exogenous inorganic compounds). These components are frequently in the form of traces, but nevertheless may have a considerable impact in the system physiology. Thus, the study of cell biochemistry in both normal and pathological/stressed situations is a key-step towards an overall understanding of cellular metabolic mechanisms. Therefore, the development of imaging and analytical techniques enabling the investigation of intracellular chemical abundances, structural organization and their related metabolic functions becomes ....

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方案

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1. Sample Holder Preparation

  1. Sample holder design and preparation
    1. Manufacture a sample holder by drilling a 5 mm x 5 mm square in a 1-mm thick PEEK frame.
    2. Clean by rinsing with ethanol 70% (v/v) and keep in sterile plates until ready to use.
      Note. A sample holder appropriate for cell culture and cell handling is required. It needs to be designed for cell culturing, in vitro observations with optical microscopy, and nuclear microprobe analysis and imaging. This holder is made of a PEEK frame13.
  2. Sample holder preparation
    1. Prepare the Formvar so....

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结果

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Cell culture and fluorescence imaging of fluorescently labeled TiO2 NPs

We designed a sample holder adapted for cell culture, cell handling as well as multimodal analysis. Specifically, it was important that the holder permitted routine optical microscopy as well as nuclear microprobe analysis and imaging. This sample holder is based on a 2-µm thick polycarbonate foil.......

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讨论

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We describe a method providing useful information beyond what is possible with other imaging techniques, especially at the subcellular level. In addition to its imaging ability, nuclear microprobe analysis also offers possibilities of quantification of chemical elements entering in the composition of a biological sample. In the present work, we studied human cell populations and focused down to the analysis of a chosen region of interest based on a single cell exposed to TiO2 NPs. Its combination with other te.......

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披露

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The authors have nothing to disclose.

致谢

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We thank Serge Borderes for directing and editing of the video. The French National Research Agency supports the research program TITANIUMS (ANR CES 2010, n° CESA 009 01). The CNRS and the European Community as an Integrating activity provided the "Support of Public and Industrial Research Using Ion Beam Technology (SPIRIT)" under the EC contract n° 227012. This work has been supported by Marie Curie Actions - Initial Training Networks (ITN) as an "Integrating Activity Supporting Postgraduate Research with Internships in Industry and Training Excellence" (SPRITE, D1.3) under EC contract no. 317169. The C'NANO Grand Sud Ouest and the Region Aquitaine support ....

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材料

本文使用的材料清单
姓名公司目录编号评论
<强>细胞培养
U2OSATCC， LGC 标准品 ATCC HTB-96
培养基 MCCOY 5A 不含 L-谷氨酰胺Dominique DUTSCHERL0211-500
FBS 500 mLDominique DUTSCHER500105U
青霉素/链霉素 ThermoFisher Scientific11548876
 L-谷氨酰胺 200 mM，100 mL Invitrogen25030024
Geneticin  20 mLThermoFisher Scientific10092772
胰蛋白酶-EDTA 0.25% （v/v）  500 mLThermoFisher Scientific11570626
Viromer RedLipocalyxVR-01LB-01
基质-roGFP 质粒AddGene#49437
Hoechst 33342ThermoFisher ScientificH3570小心处理
NPs制备
TiO2 P25 AEROXIDEDegussa/赢创
异硫氰酸四甲基罗丹明（TRITC）SIGMA-ALDRICHT3163NPs的表面改性
样品制备
聚碳酸酯箔GoodfellowCT301020
聚醚醚酮载体 （PEEK）MatechplastA-239-4047
乙醇，ACS 纯水SIGMA-ALDRICH02860-6x1L
氯仿，无水，99%SIGMA-ALDRICH372978-1L 小心 有毒
Formvar 100 g琼脂 ScientificAGR1201有害。在 10 & μ 的浓度下使用;克/mL 氯仿
NaOHSIGMA-ALDRICHS5881-500G
样品固定
粉末，95% 多聚甲醛SIGMA-ALDRICH158127-500G注意 有毒。用作 PBS
PBS（pH 7.4，不含 Ca2+ 和 Mg2+）ThermoFisher Scientific11503387
Prolong Gold 抗淬灭试剂ThermoFisher ScientificP36934
Triton X-100SIGMA-ALDRICH93443
<强>样品冷冻固定
液氮空气液体sante有害
甲基丁烷 >=99%SIGMA-ALDRICH M32631-1L注意 有毒
铝制转移板自制
蒸馏水和去离子水自制在实验室中使用 Barnstead Smart2Pure 系统生产
Parafilm VWR52858-000
设备
Barnstead Smart2PureThermoFisherScientific50129870
生物安全工作台，II 类ThermoFisher ScientificMSC-Advantage
TC20 自动细胞计数仪Biorad145-0102SP
计数玻片 2 孔Biorad1450016
PIPS 检测器，25 mm2,12 keV 能量分辨率 @5.5 MeV堪培拉 PD25-12-100AM
高分辨率 Si （Li） 固体探测器，145-eVenergy 分辨率，@Mn-K&α;牛津仪器
Everhart-Thornley 型二次电子检测器 （SED） Orsay Physics1-SED
XRF 校准标准 钠或氯作为 NaCl微物质34381
XRF 校准标准 镁作为 MgF2微量物质34382
XRF 校准标准 铝作为金属微物质34383
XRF 校准标准 硅作为 SiO微物质34384
XRF 校准标准 硫作为 CuSx微量物质34385
XRF 校准标准 钙作为 CaF2微量物质34387
XRF 校准标准 钛作为金属微量物质34388
XRF 校准标准 铁作为 Fe 金属微量物质34389
Sonicator 750WSonics 材料11743619
3MM 微探针Bioblock scientific220-05
真空冻干机ElexienceEK3147
光学显微镜 Zeiss AxioObserver Z1Carl Zeiss MicroImaging， GmbH431006-9901
电动载物台 xyCarl Zeiss MicroImaging， GmbH432031-9902
EC Plan-Neofluar 20X，NA 0.50 Ph2 M27 物镜Carl Zeiss MicroImaging， GmbH420351-9910
平场复消色差物镜 63X，NA 1,40 Ph3M27 物镜Carl Zeiss MicroImaging， GmbH420781-9910
蔡司滤光片组 02Carl Zeiss MicroImaging， GmbH488002-9901
蔡司滤光片组 38HECarl Zeiss MicroImaging， GmbH489038-9901
蔡司滤光片组 31Carl Zeiss MicroImaging， GmbH000000-1031-350
化学通风柜ErlabCaptair SD321
粒子加速器HVEEsingletron
Software
ImageJ 软件美国国立卫生研究院ImageJ 1.51
SimNRA 软件Max-Planck-Institut für Plasmaphysik，德国SIMNRA 6.06
Gupix 软件加拿大圭尔夫大学GUPIXWIN 2.2.4
的 4% 溶液有害

参考文献

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  1. Krug, H. F., Wick, P. Nanotoxicology: An Interdisciplinary Challenge. Angew. Chem. Int. Ed. 50 (6), 1260-1278 (2011).
  2. Van Hove, M. A. From surface science to nanotechnology. Catalysis Today. 113 (3-4), 133-140 (2006).
  3. Le Trequesser, Q., Seznec, H., Delville, M. H.

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