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 imaging. This holder is made of a PEEK frame13.</li>
	</ol>
	</li>
	<li>Sample holder preparation
	<ol>
		<li>Prepare the Formvar solution by dissolving 1 mg of Formvar powder in 100 mL of chloroform.</li>
		<li>Stretch the polycarbonate foil on a metallic frame so that it presents no fold.</li>
		<li>Use a sterile tip to spread the Formvar solution around the sample holder hole.</li>
		<li>Put down delicately the sample holder with glue onto the stretched polycarbonate foil (a 5 mm x 5 mm square).</li>
		<li>Repeat the previous sequence to prepare one sample holder for each replicate. 
		Note. The polycarbonate (PC) film is biocompatible, thick enough and resistant to the different protocols and analytical constraints. 
		Caution- Chloroform is toxic. Avoid inhalation, ingestion or contact with skin. Always use a properly functioning chemical fume hood and appropriate filters.</li>
	</ol>
	</li>
	<li>Sample holder sterilization
	<ol>
		<li>Place the mounted sample holder in a conical flask with 50 mL of ethanol 70% (v/v) under agitation at 180 rpm and +37 &#176;C overnight, using an incubator/agitator.</li>
		<li>Rinse the holder several times in sterile distilled water.</li>
		<li>Air dry them in sterile conditions and expose them to ultraviolet light (germicidal lamp from Biosafety bench, class II) for 30 min on each side.</li>
		<li>Keep the samples in sterile 12-well plates (one sample holder per well) until ready to use. 
		Note: Multiple sample holders can be stored at room temperature in sterile and dry 12-well plates sealed with parafilm for several days.</li>
	</ol>
	</li>
</ol>
2. Growth of Cells in the Appropriate Sample Holder.
Caution: Protocol must be carried out in a biosafety laminar flow bench (Class II) to exclude contaminating micro-organisms. Handle antibiotics (e.g. penicillin, streptomycin) with gloves. Respect best practices when handling biological materials (Cell lines, genetically modified derived human cells).
Critical: The cell lines used should be checked to ensure that they are not infected with Mycoplasma.
<ol>
	<li>Transfect U2OS cell line with plasmid bearing Matrix-roGFP 17,18,19 using a transfection method in accordance to protocol established by manufacturer.</li>
	<li>To confirm transfection with the plasmid-carrying the biosensor and to prevent plasmid loss, select and maintain the transfected cells on the appropriate culture medium.</li>
	<li>Visualize transfected cells by fluorescence microscopy to ensure the transfection has occurred and to confirm the expression of the fluorescence and the display of a reticular and tubular mitochondrial network19. 
	Pause point: Cells can be frozen in liquid nitrogen for several years.</li>
	<li>Use the transfected cell population by culturing the cells in an appropriate medium in order to obtain sub-confluent cell populations. Grow cells at 37 &#176;C, 5% (v/v) CO2 in a saturated water atmosphere.</li>
	<li>Seed cells in a concentration, such as they are at 80% confluence the day of fixation. Typically, a concentration of 500 cells per &#181;L could be used. Harvest cells using 400 &#181;L of Trypsin-EDTA 0.25% (v/v) and keep for 3 min at 37 &#176;C. Stop trypsin action with 1 mL of culture medium. Pellet the cells by centrifugation for 5 min at 230 x g and +4 &#176;C, then remove supernatant, and add the desired volume of fresh culture medium. 
	Note.The protocol is applicable for all cellular types and the number of cells seeded is dependent on the cell size and on cell doubling time. 
	Caution: Avoid subjecting trypsin, antibiotics, and serum to repeated freeze-thaw cycles. Keep them sterile. Divide the stock solution into aliquots and freeze them at &#8722;20 &#176;C. Store the aliquots until the expiration date. Carefully rinse the cell pellet in order to completely remove the trypsin. Traces of residual trypsin will delay and decrease the plating efficiency.</li>
	<li>Count the cells and perform a dilution with fresh complete culture medium kept at 37 &#176;C in order to obtain a cell suspension with 500 cells per &#181;L. 
	Note: The concentration of the cell suspension has to be adapted as a function of the cell type, in order to plate a 40 &#181;L drop.</li>
	<li>Plate a 40 &#181;L drop onto the center of the polycarbonate foil. Carefully place the sample for 2 h at +37 &#176;C, 5% (v/v) CO2 in a saturated water atmosphere. 
	Critical: Once the sample is placed in the incubator, limit as much as possible any mechanical movement to favor fast cell attachment. According to the cell type, it could be necessary to incubate cells longer than 2 h for optimal platting efficiency. Check that the incubator atmosphere is well saturated to prevent drops from evaporating.</li>
	<li>Check that cells are well-attached on the polycarbonate foil using an optical microscope. Gently add 2 mL of fresh complete medium and keep for 24 h.</li>
</ol>
3. Nanoparticles Preparation and Exposure
NOTE: Fluorescent dye-modified TiO2 NPs were designed, synthesized, and chemically modified with tetramethyl rhodamine isothiocyanate (TRITC).20,21 This surface modification allows nanoparticle detection, tracking and localization in situ and in cellulo in both living and fixed cells or in multicellular organisms.12,13,18
Caution: Nanomaterials and Nanoparticles must be handled with care. Avoid inhalation, ingestion or contact with skin. To prevent dissemination in air, nanoparticles are maintained in solution (ultrapure water).
<ol>
	<li>Prepare a suspension of 1 mg.mL&#8722;1 ofTiO2 NPs in ultrapure water. Disperse the TiO2 NPs using intense 1-min sonication pulses at RT (750 W, 20 kHz, amplitude: 30%) using an ultra-sound generator and a dedicated conical microprobe.</li>
	<li>Dilute TiO2 NPs at the desired concentration in the culture medium in order to obtain an exposure suspension of 4 &#181;g.cm&#8722;2 (final concentration). Replace medium with the appropriate volume of medium containing NPs on cells and mix gently for homogeneous distribution of the TiO2 NPs. Prepare control cells similarly without the addition of TiO2 NPs.</li>
	<li>Incubate the cell populations for 24 h at 37 &#176;C, 5% (v/v) CO2 and a saturated water atmosphere.</li>
</ol>
4. Paraformaldehyde fixation and fluorescence microscopy.
<ol>
	<li>Prepare a fresh solution of paraformaldehyde solution (PFA, 4% w/v) buffered in Phosphate Buffer Saline (PBS, pH 7.4).
	<ol>
		<li>Dissolve 4 g of PFA powder in 100 mL of PBS. Heat the solution to 65 &#176;C while stirring using a magnet and a magnetic stirrer in a fume hood. Increase the pH by adding 1 M NaOH one drop at a time until the solution clears. Cool the solution to room temperature and adjust the pH to 7.4. Use the freshly prepared solution immediately or keep at +4 &#176;C and protect from light. 
		Note: Pre-made PFA solutions could be used however an extemporaneous preparation of PFA guarantees better fixation quality and long-term conservation of the fixed samples. 
		Caution: PFA is toxic; avoid inhalation, ingestion or contact with skin. Always use a properly functioning chemical fume hood and appropriate filters.</li>
	</ol>
	</li>
	<li>Once, the incubation time has elapsed, remove the cell culture medium containing the TiO2 NPs. Rinse the cell populations once with fresh culture medium and once with PBS (2 mL). Remove PBS, rinse quickly with an aliquot of fresh and cold PFA (4% w/v, +4 &#176;C).
	<ol>
		<li>Add PFA (2 mL, 4% w/v, +4 &#176;C). Incubate 15 min at room temperature.</li>
		<li>Remove the PFA, rinse the cells with PBS (2 mL, 3 times, 5 min) under agitation. 
		Pause point: These chemically fixed samples can be used for the &#34;plunge-freeze&#34; procedure after fluorescence imaging (go to step 5) or used only for fluorescence imaging (go to step 4.3).</li>
	</ol>
	</li>
	<li>Stain the nucleus with Hoechst33342 incubating the cells for 10 min in PBS. Hoescht33342 is used at a final concentration of 500 nM. After incubation remove the solution and rinse with PBS. 
	Caution: Hoechst33342 is cell permeant and may be toxic. Avoid direct contact, and use gloves while preparing and using Hoechst33342. 
	Critical: Ensure that the Hoechst33342 staining solution is freshly prepared and maintained in sterile conditions.</li>
	<li>Process fixed samples for in situ and single cell imaging using fluorescence microscopy (Figure 1).</li>
</ol>
5. &#34;Plunge-Freezing&#34; Fixation and Dehydration
<ol>
	<li>Prepare an aluminum transfer plate by cooling it in liquid nitrogen. Store the plate into a box filled with liquid nitrogen and maintain the plate surface above the liquid in the cold nitrogen vapor. 
	Note: The transfer plate can be made of any metallic plate (here, we used aluminum) that could be cooled down to liquid nitrogen temperature, and that can enter the freeze-dryer sample chamber. 
	Critical: The box should be kept closed as much as possible in order to prevent water vapor deposition onto the cold plate surface. The sample stored on the plate during preparation should not be covered by liquid nitrogen.</li>
	<li>Rinse cells once in culture medium and then twice more, briefly, in sterile and ultrapure water to get rid of any trace of remaining extracellular salts. 
	Critical: Ensure that the media are freshly prepared and maintained in sterile conditions. Heat all the media at 37 &#176;C before being used. The rinse must be very brief (few seconds) and the excess of liquid on the samples removed as fast as possible.</li>
	<li>Plunge-freeze the cells at -150 &#176;C in liquid nitrogen-chilled 2-methylbutane during 30 s and place them onto the aluminum transfer plate. Store samples here during the preparation of all other samples. When samples are all cryofixed, transfer all-at-once by placing the transfer plate in a freeze-dryer. 
	Critical: The overall cryofixation process should not last more than 20 min in order to prevent structural modifications to appear in samples temporarily stored in the transfer box.</li>
	<li>Freeze-dry the samples using the following sequences: 1) perform a primary desiccation for 12 to 24 h (-99 &#176;C, 10-3 mbar) at low pressure and low temperature, then 2) perform a secondary desiccation phase for at least 24 h, increasing the plate temperature to +40 &#176;C while keeping the pressure low (+40 &#176;C, 10-3 mbar). 
	Caution: Liquid nitrogen is extremely cold and can cause severe frostbite or eye damage upon contact. Use adapted bench top containers for transport and wear safety equipment (cryogenic gloves, eye and face protection). 
	Caution: 2-Methylbutane is extremely flammable. A harmful contamination of the air can be reached rather quickly on evaporation of this substance at +20 &#176;C. Avoid inhalation, ingestion or contact with skin. Use breathing and eye protection, and protective gloves. Always use a properly functioning chemical fume hood. May be stored at +4 &#176;C. 
	Critical: Use an appropriate thermometer (-150 &#176;C) to monitor the 2-Methylbutane temperatures during the &#34;Plunge-freeze&#34; session. The 2-methylbutane must be kept cool in a liquid phase. Transferring cryofixed samples to the ambient atmosphere may cause cellular damage due to the temperature increase or water vapor condensing on the sample surface. Accordingly, the transfer should be made as fast as reasonably possible (30 s) 
	Pause point: After cryofixation, samples can be stored at room temperature for several days in sterile and dry conditions (protected from dust and moisture). Carefully handle the samples with fine forceps and place them in a sterile 12-well plate sealed with parafilm. Desiccant can be used to dry the atmosphere.</li>
</ol>
6. Nuclear Microprobe Analysis
NOTE: Nuclear Microprobe Analysis was carried out at the microbeam line of AIFIRA (Applications Interdisciplinaires des Faisceaux d&#39;Ions en R&#233;gion Aquitaine) using the complementary ion beam analytical techniques Particle Induced X-ray Emission (&#181;-PIXE) and Scanning Transmission Ion Microscopy (&#181;-STIM). The facility is based on a 3.5 MV particle accelerator delivering light ion beams in the MeV energy range.22,23
Pause point: AIFIRA is an ion beam facility hosted by the University of Bordeaux that offers an access to national and international teams after scientific evaluation of the proposed experiment.
<ol>
	<li>Mount the PEEK sample holders on an apertured plate using double-sided tape.</li>
	<li>Place the plate supporting sample in a vertical plane perpendicular to beam direction.</li>
	<li>Close the analysis chamber and vacuum the analysis chamber.</li>
	<li>Scanning Transmission Ion Microscopy (&#181;-STIM) 
	NOTE: STIM is used to record areal density maps of cells after conversion of energy loss to cellular mass, taking advantage of the fact that the energy loss is proportional to the sample areal density (expressed in &#181;g.cm-2).
	<ol>
		<li>Use a 2 MeV Helium (He+) microbeam as a probe with a size in the focal plane of around 300 nm in diameter and at low fluency (2000 ions.s-1). Measure the energy of the transmitted ions with a planar silicon detector (PIPS detector, 25 mm2, 11 keV energy resolution @ 5.4 MeV), placed behind the sample in the beam axis.</li>
	</ol>
	</li>
	<li>Particle induced X-ray Emission (&#181;-PIXE) and Rutherford Backscattering Spectrometry (&#181;-RBS). 
	NOTE: &#181;-Pixe and &#181;-RBS analyses provide the spatial distribution and quantification of chemical elements with a sub-micrometer resolution.
	<ol>
		<li>Use a 1.5 MeV proton (H+) microbeam (50-150 pA), focused down to a diameter of 1 &#181;m and scan over the same cells of interest spotted by STIM from 4 to 8 hours and around 100 x 100 &#181;m2.</li>
		<li>Collect induced X-ray photons emitted from atoms present in the sample (heavier than Na) by a high-resolution Si(Li) solid-state detector (145 eV energy resolution, @Mn-K&#945;) positioned at 45&#176; from incoming beam axis. Use the detected photon energy to identify the nature of emitters (e.g. Z of element) and X-ray intensity to determine the element concentration.</li>
		<li>Simultaneously collect back-scattered protons at -135&#176; with a silicon detector (partially depleted detector, 25 mm2, 11 keV FWHM @ 5.4 MeV) to measure the total number of incoming particles and to normalize X-ray intensities. 
		Critical: A collection of certified calibration standards for atomic concentration quantification is used in order to calibrate the X-ray detector response. Reference materials are a collection of thin atomic films deposited on 6.3 &#181;m thick Mylar foils. Emitted X-Ray energies range from 0.6 (Li, K line) to 20.2 keV (Rh, K line).</li>
	</ol>
	</li>
</ol>
7. Data analysis
<ol>
	<li>Reconstruct chemical elemental maps using the IBA-J plugin for ImageJ.24 
	NOTE: Dedicated software in the form of a plugin for ImageJ has been developed to process raw nuclear microprobe analysis data. Raw data correspond to a list of events arising from particle beam interaction with cells that are recorded along with the beam position as a chronological sequence.
	<ol>
		<li>Use the IBA-J plugin to sort events according to their type: transmitted ion energy (STIM), backscattered ion energy (RBS) or X-ray photon energy (PIXE). Then, process separately each type of data.</li>
		<li>Calculate STIM area density map</li>
		<li>Calculate average X-ray photon energy spectra and define for each chemical element of interest an energy window in order to retrieve the element spatial distribution.</li>
		<li>Define regions of interest (ROI) (e.g. individual cells, dense structure, aggregates, etc.) and calculate the corresponding x-ray spectra.</li>
	</ol>
	</li>
	<li>Analyze the corresponding RBS spectra in order to calculate the total number of incident particles25.</li>
	<li>Fit X-ray spectra using the Gupix software26 in order to determine the element concentration for each ROI. 
	NOTE: Typical limit of detection (LOD) for the method is in the range 1 to 10 &#181;g.g-1 in dry mass depending on the atomic number of elements (LOD is decreasing with Z).</li>
</ol>

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The authors have nothing to disclose.

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><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

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. ...

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

Research

JoVE Journal

Chemistry

7.1K Views.  Université de Bordeaux. Beschrijven we een procedure voor de opsporing van chemische elementen aanwezig in situ in menselijke cellen, alsmede hun kwantificering in vitro . De methode is geschikt voor elk celtype en is vooral nuttig voor de kwantitatieve chemische analyses in afzonderlijke cellen na in vitro metaaloxide nanodeeltjes blootstelling.

Video: 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.

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

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.

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

Engineering

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.

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.

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

Cancer Research

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.

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.

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.

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

Biochemistry

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.

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.

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.

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

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

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