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&#176; 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&#176; 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 the research program TOX-NANO (n&#176;20111201003) and the research program POPRA (n&#176; 14006636-034).

1. Sample Holder Preparation
<ol>
	<li>Sample holder design and preparation
	<ol>
		<li>Manufacture a sample holder by drilling a 5 mm x 5 mm square in a 1-mm thick PEEK frame.</li>
		<li>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 imagin...

<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>

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...

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 necessary. Very few methods are able to provide an in situ quantitative piece of information concerning the overall chemical nature of a given sample. Apart from methods analyzing samples in the bulk form, in situ analyses consider biological samples in their integrality without losing mass and structural information, thereby preserving their constituent chemicals (trace elements and ions) and proteins. Furthermore, as the nanosciences continue to develop, improved imaging and analytical methods for environmental monitoring at the cellular scale will be necessary to observe and quantify nano-object behaviors and interactions.1
Nanoparticles (NPs) have been defined as objects exhibiting at least one facial dimension in the range 1 and 100 nm.2 Due to their particular physicochemical properties, NPs are extensively used in industry. NPs are employed in bio-applications and in nanomedicine.3,4 Despite the numerous physicochemical characteristics of NPs, they may generate some risks of adverse effects on human health and environment. These risks can be induced by both prolonged and repetitive exposures at various concentration levels and this has not yet been clearly established.5,6,7,8 In particular, the fate of NPs inside cells and the associated cellular responses are, to date, not fully described. This is in part due to the scarcity of methods that allow the detection and quantification of internalized NPs in a single cell.9
The classical analytical tools used to estimate the cellular dose of nanoparticles are microscopies, mass spectrometry (MS), inductively coupled plasma MS (ICP-MS)10,11 and liquid chromatography MS (LC-MS), but they only provide useful information at the macroscopic scale. None of them can provide a precise evaluation of the subcellular NPs content nor the NPs distribution without the use of fractionation methods. A systematic assessment of the dose-response is thus impossible with these methods, as opposed to methods based on atomic spectroscopy such as nuclear microprobe analysis12,13, synchrotron X-ray fluorescence microscopy14, and Secondary Ion Mass Spectrometry (SIMS).15,16 These methods are particularly interesting as they complement observations made using fluorescence microscopy, especially when NPs cannot be labeled with fluorescent molecules and are thus studied in their native state. To some extent, even when NPs are grafted with fluorophores, (i) quantification remains difficult because the tagging level per NP is unknown and (ii) the chemical modification of the NP surface may modify its cellular distribution.
In this article, we focus on a method based on a combination of nuclear microprobe techniques aiming at imaging the morphology and elemental composition of biological specimens in major, minor, and trace concentrations.
Nuclear microprobe analysis proves to be particularly suitable for the measurement of trace chemical elements in biological tissues. Both the beam lateral resolution (0.3 to 1 &#181;m) and sensitivity in chemical element detection (from 1 to 10 &#181;g.g-1 dry mass) are well suited for studies at the cellular level. Nuclear microprobe techniques are based on particles detection (photons, electrons, ions) emitted after the ion beam (typically running at MeV energies) interacts with atoms present in the sample. Interactions occurring in cells are mainly: 1) excitation/ionization of atoms followed by an emission of photons after atoms return to their fundamental state; and 2) diffusion of incoming particles leading to change in their energy and direction. The measurement of emitted particle energy allowsthe identification of atoms involved in the interaction. To perform mapping of elements, the ion microbeam is repeatedly scanned over the sample surface, often over an area of about 100 by 100 &#181;m2 containing several cells. Emitted particles are detected and their energy is recorded for each beam position. Sorting of particles according to the beam position, thus identifying the structure responsible for the emission of such particles is the aim of data treatment. Here, we precisely describe an approach based on fluorescence microscopy and nuclear microprobe analysis to detect as well as to quantify exogenous NPs at the cellular and sub-cellular scales, in order to investigate the consequences of NP interactions with living systems. We shall particularly focus on the opportunities offered by this method in terms of in situ quantification of titanium dioxide nanoparticles (TiO2 NPs) aggregates at the subcellular level.

<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

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 &#181;g.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.

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-&#181;m thick polycarbonate...

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

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.

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

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

Nanoparticles are increasingly used in industry and medicine due to their unique physiochemical properties. However, concerns for human health associated with prolonged nanoparticle exposure persists. These risks can be quantified by studying the behavior of nanoparticles inside cells and the induced metabolic responses of cells to nanoparticles.This is partially due to the lack of methods that allow the detection and quantification of internalized nanoparticles in a sincle cell. While numerous analytical tools, including microscopy and mass spectrometry, can be used to estimate the cellular uptake of nanoparticles, these only provide qualitative information at a microscopic level. On the contrary, in situ methods based on atomic spectroscopy can reduce the quantity of imaging artifacts that arise from sample preparation and can yield direct observation of nanoparticles.To take advantage of this, we present a correlative approach which allows us to study nanoparticles either in the native state or lab bed with a fluorescent tag. Here, we describe a method based on the combination of fluorescence microscopy and nuclear microprobe analysis. This provides images of the cellular density, chemical element special distribution, and the amount of nanoparticles per cell.As a demonstration, we shall research from cells exposed to titanium dioxide nanoparticles over 24 hours using both fluorescence microscopy and nuclear microprobe analysis. For this protocol, a custom sample holder made with the polymer PEEK is needed. It must be suited for cell culturing and in vitro observations.Cover the holder with two micron thick polycarbonate foil. The polycarbonate foil is glued to the PEEK dish with a thin layer of Formvar solution. Once mounted, the sample holder needs to be sterilized.Multiple sterile sample holders may be stored, one per well, in a sterile twelve-well plate until they are needed for use. Cells transfected with matrix row GFP plasmids are checked by fluorescence microscopy to insure transfection has occurred successfully, and that they express the fluorescent protein. Harvest the cells with trypsin, and incubate them for three minutes at 37 degrees Celsius.Stop the trypsin action by adding fresh culture medium. Pellet the cells by centrifugation for five minutes at 1200 revolutions per minute at four degrees Celsius. Remove the supernatant and add an appropriate volume of fresh culture medium.Count the cells and perform a dilution with fresh and thermostated complete culture medium to obtain a cell suspension of 500 cells per microliter. Plate a 40-microliter drop at the center of the polycarbonate foil. Carefully place the sample in the cell incubator for two hours.Gently add two milliliters of fresh culture medium and leave them for 24 hours. Fluorescent dimodified titanium oxide nanoparticles were designed, synthesized, and grafted with commonly used fluorophores in order to detect, track, and localize nanoparticles in vitro. For this step, a titanium dioxide nanoparticle suspension in ultra-pure water with a concentration of one milligram per milliliter should already have been prepared.Disperse the nanoparticles by using one-minute long intense sonication pulses at room temperature. Dilute the nanoparticles in an appropriate culture medium to obtain an exposure suspension of four micrograms per square centimeter. Switch the previous cell culture medium to this new nanoparticle-containing medium and mix it gently to achieve a homogeneous nanoparticle distribution.Prepare a set of control cells in the same manner without the addition of titanium dioxide nanoparticles. Incubate the cell populations for 24 hours. Using paraformaldehyde, these samples may be fixed and processed for in situ and single-cell imaging using fluorescence microscopy.Cryogenic physical fixation, however, preserves the cell ultrastructure and biochemical integrity. Plunge freezing fixation rapidly stops the cellular activity within milliseconds and avoids using fixative compounds. To cryogenically fix the cells, we perform the following.Prepare an aluminum transfer plate by cooling it in liquid nitrogen. Store the plate in a box filled with liquid nitrogen, keeping the plate surface above the liquid in the cold nitrogen vapor. Chill a sufficient amount of 2-methylbutane to 150 degrees Celsius.Prepare one 50 milliliter Falcon with culture medium and two Falcons with ultra-pure water. Ensure absorbent paper is nearby to dry the sample before freezing it. Rinse cells once in culture medium and then two more times in sterile, ultra-pure water to remove excess salts remaining in the culture medium.Quickly dry the sample on the absorbent paper. Plunge-freeze the cells in the chilled 2-methylbutane for 30 seconds and place them on the chilled aluminum transfer plate. It is critical that the box be kept closed as much as possible in order to prevent water vapor from condensing on the cold plate surface.After freezing all samples, freeze-drying is performed by the following method to sublimate away any water. First, perform a primary desiccation for 12 to 24 hours at low pressure and low temperature. Next, keeping the pressure low, perform a secondary desiccation phase for at least 24 hours with the plate temperature increased to 40 degrees Celsius.After cryofixation, samples can be stored at room temperature for several days in sterile and dry conditions protected from dust and moisture. Nuclear microprobe analysis was conducted at the Ifera facility in Bordeaux, France. A 3.5 megavolt singletron particle accelerator delivers a light ion beam in the megaelectron volt energy range.Chemical element imaging was carried out on the microprobe beamline using complementary ion beam analytical techniques. The techniques used through a micro-PIXE, particle-induced X-ray emission, STIM, scanning transmission ion microscopy, and micro-RBS, rutherford backscattering spectrometry. Samples are positioned inside the analysis chamber and irradiated under vacuum with different particles in order to perform different ion beam analyses.STIM is used to record aerial density maps of cells based on the energy particles loose passing through regions of cells with different densities. Micro-PIXE and micro-RBS analysis provide the spatial distribution and quantification of chemical elements at a single-cell level. A 1.5 megaelectron volt proton microbeam is focused down to a diameter of about 1 micrometer and it is scanned across the cells of interest, identified by STIM.X-rays emitted from atoms present in the sample, ranging from sodium to titanium, permit the elemental concentrations to be determined. Backscattered protons are collected in order to measure the total number of incoming particles required to normalize X-ray intensities. It is critical to have certified calibration standards for the quantification of elemental concentrations in order to calibrate the X-ray detector response.In order to the analyze the ion beam data, we developed a new plugin for ImageJ. First, STIM density maps were calculated. Contrast in scanning transmission iron microscopy images is due to local differences in density and allows the detection of cell structures such as the nucleus and cytoplasm.Several maps can be processed at once. Each map corresponds to the medium transmitted energy from incoming particles. The second step in data analysis consists in calculating individual cell spectra.Particle-induced X-ray emission analysis yields both the chemical composition of the sample and the elemental maps of chemical elements. Chemical element maps are computed after sorting photons according to the beam position at the time of recording and selecting an energy window centered around a specific element. Maps usually represent the number of detected events at the beam position and contain quantitative data.A stack of chemical maps, one for each selected element, is calculated. Regions of interest for each cell are found using the PIXE data for phosphorus. The spectra corresponding to each whole cell is then calculated by summing the individual spectra across each cell's region of interest.From here, quantitative data about chemical abundances in each cell can be extracted using dedicated PIXE and IBS analysis software. Here we show fluorescence imagery of cells after paraformaldehyde fixation. The top row shows cells that have not been exposed to titanium dioxide nanoparticles while the bottom row has been.The blue channel shows the cell nucleus, the green channel shows mitochondria, and the red channel shows titanium. It can be clearly seen that no titanium dioxide is present in the controls. From the merged channel at the right, it can be seen that nanoparticles are found exclusively in the cytoplasm and are excluded from mitochondria.What this method lacks, however, is quantitative information about the nanoparticle concentration. Nuclear microprobe analysis can hope to provide this. These images have all been taken using various microprobe analysis techniques.Again, the top row shows the control cells and the bottom row shows cells containing titanium nanoparticles. The leftmost image shows STIM measurements of the density of the cryogenically fixed cells. The following three panels show the abundance of potassium, phosphorus, and titanium present in the cells obtained by PIXE.Again, no titanium is visant in the controls nor in the cell nuclei. In order to quantify, cell by cell, the uptake of titanium, we define regions of interest based on the cell boundaries shown in the rightmost panel and measure the elemental abundances in these regions. From these PIXE measurements, we have plotted the distribution of elemental abundances at the single-cell level for the control and the exposed populations.The control population is plotted in white, and the exposed population is plotted in yellow. The median content of titanium present here is quite low compared to the four microgram per square centimeter exposure dose. The range of titanium uptake shows a large variation across the population, from 0.2 micrograms per square centimeter up to 1.8 micrograms per square centimeter.We also noticed an increase of free intracellular ions such as potassium and calcium in nanoparticle-exposed samples, suggesting an alteration to cellular homeostasis induced by the presence of titanium dioxide nanoparticles. Nuclear microprobe analysis can provide useful information on the subcellular level and provides a quantification of the chemical elements that make up a biological sample. These techniques present many advantages.One, in sample preparation which does not require chemical fixation. Two, the possibility to study larger areas with the ability to focus on additional regions of interest. Three, the quantification of chemical elements with a sensitivity of a few micrograms per gram.And four, the identification of cell compartments such as the nucleus, nucleolus, and cytoplasm. The accurate determination of those, when studying the internalization of nanoparticles in single cells, is essential for quantitative nanoparticle toxicology. As shown in the cases studied here, the ability to observe and quantify nanoparticles within individual cells allow us to better understand the value accumulation of endogenous and exogenous chemical elements, such metal oxide nanoparticles.This protocol highlights the suitability of nuclear microprobe analysis for future studies of nanoparticle interaction with living cells. The quantitative approach gives information about the impact of these nanoparticles in terms of detection, identification, localization, and quantification, and in single-cell level for both native and chemical modified-nanoparticles.

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Research

JoVE Journal

Chemistry

7.0K vistas.  Université de Bordeaux. Nanoparticles are increasingly used in industry and medicine due to their unique physiochemical properties. However, concerns for human health associated with prolonged nanoparticle exposure persists. These risks can be quantified by studying the behavior of nanoparticles inside cells and the induced metabolic responses of cells to nanoparticles.This is partially due to the lack of methods that allow the detection and quantification of internalized nanoparticles in a sincle cell. While...