The authors gratefully acknowledge financial support by the Labex-Imust.

1. Biological Sample Preparation
All the experiments described in this study were approved by the Animal Care and Use Committee of the CECCAPP (Lyon, France) (authorization #LYONSUD_2012_004), and the experiments were carried out under the supervision of authorized individuals (L. Sancey, DDPP authorization #38 05 32).
<ol>
	<li>Add 1 ml of H2O to&#160;100 &#181;mol of Gadolinium (Gd)-based nanoparticles, wait 15 min, and add 20 &#181;l of HEPES 50 mM,&#160;NaCl 1...

<ol>
	<li>Smith, M. A., Harris, P. L., Sayre, L. M., Perry, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+accumulation+in+Alzheimer+disease+is+a+source+of+redox-generated+free+radicals.">Iron accumulation in Alzheimer disease is a source of redox-generated free radicals.</a> Proceedings of the National Academy of Sciences of the United States of America. 94, 9866-9868 (1997).</li><li>Wang, Y., Zhu, S., Weisman, G. A., Gitlin, J. D., Petris, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+knockout+of+the+Menkes+disease+copper+transporter+demonstrates+its+critical+role+in+embryogenesis.">Conditional knockout of the Menkes disease copper transporter demonstrates its critical role in embryogenesis.</a> PloS one. 7, (2012).</li><li>Reske-Nielson, E., Lou, H. O., Andersen, P., Vagn-Hansen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain-copper+concentration+in+Menkes'+disease.">Brain-copper concentration in Menkes' disease.</a> Lancet. 1, 613 (1973).</li><li>Hayashi, H., 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=Various+copper+and+iron+overload+patterns+in+the+livers+of+patients+with+Wilson+disease+and+idiopathic+copper+toxicosis.">Various copper and iron overload patterns in the livers of patients with Wilson disease and idiopathic copper toxicosis.</a> Medical molecular morphology. 46, (2013).</li><li>Lobinski, R., Moulin, C., Ortega, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+speciation+of+trace+elements+in+biological+environment.">Imaging and speciation of trace elements in biological environment.</a> Biochimie. 88, 1591-1604 (2006).</li><li>Dev&#232;s, G., Bouhacina, T., Ortega, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STIM+mass+measurements+for+quantitative+trace+element+analysis+within+biological+samples+and+validation+using+AFM+thickness+measurements.">STIM mass measurements for quantitative trace element analysis within biological samples and validation using AFM thickness measurements.</a> Spectrochimica Acta B. 59, 1733-1738 (2004).</li><li>Twining, B. 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=Quantifying+trace+elements+in+individual+aquatic+protist+cells+with+a+synchrotron+X-ray+fluorescence+microprobe.">Quantifying trace elements in individual aquatic protist cells with a synchrotron X-ray fluorescence microprobe.</a> Analytical chemistry. 75, 3806-3816 (2003).</li><li>Guerquin-Kern, J. L., Wu, T. D., Quintana, C., Croisy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+analytical+imaging+of+the+cell+by+dynamic+secondary+ion+mass+spectrometry+(SIMS+microscopy).">Progress in analytical imaging of the cell by dynamic secondary ion mass spectrometry (SIMS microscopy).</a> Biochimica et biophysica acta. 1724, 228-238 (2005).</li><li>Binet, M. R., Ma, R., McLeod, C. W., Poole, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+characterization+of+zinc-+and+cadmium-binding+proteins+in+Escherichia+coli+by+gel+electrophoresis+and+laser+ablation-inductively+coupled+plasma-mass+spectrometry.">Detection and characterization of zinc- and cadmium-binding proteins in Escherichia coli by gel electrophoresis and laser ablation-inductively coupled plasma-mass spectrometry.</a> Analytical biochemistry. 318, 30-38 (2003).</li><li>Becker, J., Gorbunoff, A., Zoriy, M., Izmer, A., Kayser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+near-field+laser+ablation+inductively+coupled+plasma+mass+spectrometry+(NF-LA-ICP-MS)+at+nanometre+scale+for+elemental+and+isotopic+analysis+on+gels+and+biological+samples.">Evidence of near-field laser ablation inductively coupled plasma mass spectrometry (NF-LA-ICP-MS) at nanometre scale for elemental and isotopic analysis on gels and biological samples.</a> J. Anal. Atom. Spectrom. 21, 19-25 (2006).</li><li>Seeley, E. H., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+imaging+by+mass+spectrometry:+a+new+frontier.">3D imaging by mass spectrometry: a new frontier.</a> Analytical chemistry. 84, 2105-2110 (2012).</li><li>Pornwilard, M. -. M., Weiskirchen, R., Gassler, N., Bosserhoff, A. K., Becker, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+bioimaging+techniques+of+metals+by+laser+ablation+inductively+coupled+plasma+mass+spectrometry+for+diagnosis+of+fibrotic+and+cirrhotic+liver+disorders.">Novel bioimaging techniques of metals by laser ablation inductively coupled plasma mass spectrometry for diagnosis of fibrotic and cirrhotic liver disorders.</a> PloS one. 8, (2013).</li><li>Cremers, D. A., Radziemski, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of laser-induced breakdown spectroscopy. , (2006).</li><li>Miziolek, A. W., Palleschi, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laser-Induced Breakdown Spectroscopy: Fundamentals and Applications. , (2006).</li><li>Motto-Ros, V., 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=Mapping+nanoparticles+injected+into+a+biological+tissue+using+laser-induced+breakdown+spectroscopy.">Mapping nanoparticles injected into a biological tissue using laser-induced breakdown spectroscopy.</a> Spectrochimica Acta Part B. , (2013).</li><li>Motto-Ros, V., 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=Mapping+of+native+inorganic+elements+and+injected+nanoparticles+in+a+biological+organ+with+laser-induced+plasma.">Mapping of native inorganic elements and injected nanoparticles in a biological organ with laser-induced plasma.</a> Applied Physics Letters. 101, (2012).</li><li>Mignot, 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=A+top-down+synthesis+route+to+ultrasmall+multifunctional+Gd-based+silica+nanoparticles+for+theranostic+applications.">A top-down synthesis route to ultrasmall multifunctional Gd-based silica nanoparticles for theranostic applications.</a> Chemistry. 19, 6122-6136 (2013).</li><li>Lux, F., 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=Ultrasmall+rigid+particles+as+multimodal+probes+for+medical+applications.">Ultrasmall rigid particles as multimodal probes for medical applications.</a> Angew Chem Int Ed Engl. 50, 12299-12303 (2011).</li></ol>

Applied to biological sample, this technique allows the chemical imaging, i.e. the mapping and quantification, of Gd and Si from injected Gd-based nanoparticles in different organs. From the main critical settings, the control of laser properties (wavelength, pulse energy, focusing, and stability) is critical for a precise and fine tissue ablation (i.e. mapping resolution) as well as for sensitivity. Working at high energy provides a better sensitivity but unfortunately generates degraded spatial resolu...

The wide development of nanoparticles for biological applications urged the parallel improvement of analytical techniques for their quantification and imaging in biological samples. Usually the detection and the mapping of the nanoparticles in organs are made by fluorescence or confocal microscopy. Unfortunately these methods require the labeling of the nanoparticles by a near infrared dye that can modify the biodistribution of the nanoparticles, especially for very small nanoparticles due to its hydrophobic properties. The detection of labeled nanoparticles, and especially the very small nanoparticles (size &#60; 10 nm), might thus interfere with their biodistribution at the whole body scale but also at the tissue and cell levels. The development of new devices able to detect nanoparticles without any labeling offers new possibilities for the study of their behavior and kinetics. Moreover, the role of trace elements such as iron and copper in brain illnesses and neurodegenerative diseases such as Alzheimer1, Menkes2,3, or Wilson4 suggest the interest to study and localize these elements in tissues.
Various techniques have been used to provide elemental mapping or microanalysis of different materials. In their review paper published in 2006, R. Lobinski et al. provided an overview of available standard techniques for elemental microanalysis in biological environment, one of the most challenging environments for analytical sciences5. The electron microprobe, which consists of energy dispersive X-ray microanalysis in a transmission electron microscope, can be applied to numerous studies if the element concentration is sufficient (&#62;100&#8211;1,000 &#956;g/g). To reach lower detection limits, the following techniques have been used:
<ul>
	<li>ion beam microprobe using particle induced X-ray emission &#956;-PIXE (1&#8211;10 &#956;g/g)6</li>
	<li>synchrotron radiation microanalysis &#956;-SXRF (0.1&#8211;1 &#956;g/g)7</li>
	<li>secondary ion mass spectrometry SIMS (0.1 &#956;g/g)8</li>
	<li>laser ablation inductively coupled mass spectrometry LA-ICP-MS (down to 0.01 &#956;g/g)9,10</li>
</ul>
The above-mentioned techniques provide micrometric resolution as shown in the Table 1 extracted from Lobinski et al.
3D reconstruction of serial 2D investigations could also be proposed for the reconstruction of deeper tissues11. However, all devices and systems require both qualified professionals, moderate to highly expensive equipment and long-lasting experiments (typically more than 4 hr&#160;for a 100 &#181;m x 100 &#181;m for &#181;-SXRF and 10 mm x 10 mm for LA-ICP-MS)12. Altogether, these requirements make elemental microanalysis very restricting and incompatible with conventional optical imaging systems, fluorescence microscopy or nonlinear microscopy. Another point that we can mention here is that the quantitative measurement capability is still quite limited and depends on the availability of matrix-matched laboratory standards. The further generalization of the use of elemental microanalysis in industry processes, geology, biology and other domains of applications will generate significant conceptual and technological breakthroughs.
The purpose of the present manuscript is to propose solutions for quantitative elemental mapping (or elemental microanalysis) in biological tissues with a tabletop instrumentation fully compatible with conventional optical microscopy. Our approach is based on the laser-induced breakdown spectroscopy (LIBS technology). In LIBS, a laser pulse is focused on the sample of interest to create the breakdown and spark of the material. The atomic radiation emitted in the plasma is subsequently analyzed by a spectrometer and the elemental concentrations can be retrieved with calibration measurements performed beforehand13,14. The advantages of LIBS include sensitivity (&#181;g/g for almost all the elements), compactness, very basic sample preparation, absence of contact with the sample, instantaneous response and precisely localized (micro) surface analysis. However, the application of tissue chemical imaging remains challenging since the laser ablation of tissue must be finely controlled to perform maps with high spatial resolution together with sensitivity in the &#181;g/g range15,16.
With such solution, the adjunction of tracers or labeling agents is not needed, which allows detecting inorganic elements directly in their native environment in biological tissues. The LIBS instrument developed in our laboratory offers a current resolution inferior to 100 &#181;m with an estimated sensitivity for Gd below 35 &#181;g/g, equivalent to 0.1 mM 16, which allows the mapping of large samples (&#62;1 cm2) within 30 min. In addition, homemade software facilitates the acquisition and exploitation of the data. This instrument is used to detect, map, and quantify the tissue distribution of gadolinium (Gd)-based nanoparticles17-18 in kidneys and tumor samples from small animals, 1 to 24 hr&#160;after intravenous injection of the particles (size &#60;5 nm). Inorganic elements, which are intrinsically contained in a biological tissue, such as Fe, Ca, Na, and P, have also been detected and imaged.

<table border="0" cellpadding="0" cellspacing="0" width="747">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Laser nanosecond Nd:YAG</td>
			<td style="width:129px;">Quantel</td>
			<td style="width:119px;">Brillant</td>
			<td style="width:283px;">5ns pulse witdh, wavelength 1064 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Spectrometer</td>
			<td>Andor Technology</td>
			<td>Shamrock 303</td>
			<td>with 1200 l/mm blazed at 300 nm grating</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Detector ICCD</td>
			<td>Andor Technology</td>
			<td style="width:119px;">Istar</td>
			<td>2 ns temporal resolution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LIBS Unit</td>
			<td style="width:129px;">ILM</td>
			<td style="width:119px;"></td>
			<td>Homemade Instrumentation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:129px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gd-based nanoparticles</td>
			<td style="width:129px;">Nano-H</td>
			<td style="width:119px;"></td>
			<td>particles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES</td>
			<td style="width:129px;">Sigma-Aldrich</td>
			<td style="width:119px;">H4034</td>
			<td>for particle&#39;s dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CaCl2</td>
			<td style="width:129px;">Sigma-Aldrich</td>
			<td style="width:119px;">21108</td>
			<td>for particle&#39;s dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:129px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5886</td>
			<td>for particle&#39;s dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">mice</td>
			<td style="width:129px;">Charles River</td>
			<td style="width:119px;">depending of animal breeding</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">isoflurane</td>
			<td style="width:129px;">Coveto / Virbac</td>
			<td style="width:119px;"></td>
			<td>for anaesthesia - Isofluranum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">isopentane</td>
			<td style="width:129px;">Sigma-Aldrich</td>
			<td style="width:119px;">59060</td>
			<td>to froze the sample&#160; slowly</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">liquide nitrogen</td>
			<td style="width:129px;">Air Liquide</td>
			<td style="width:119px;"></td>
			<td>to cool down the isopentane</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">cryostat</td>
			<td style="width:129px;">Leica</td>
			<td style="width:119px;">CM-3050S</td>
			<td>to slide the samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">petri dishes</td>
			<td style="width:129px;">Dutscher</td>
			<td style="width:119px;">353004</td>
			<td>to stick the sample</td>
		</tr>
	</tbody>
</table>

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.

As shown in Figure 1, the beam of a Nd:YAG laser in the fundamental wavelength of 1,064 nm was focused vertically down on the tissue slice by a quartz lens of 50 mm focal distance. The pulse energy was 4 mJ and the repetition rate 10 Hz. In order to avoid the generation of plasma in air, the laser beam was focused around 100 &#181;m under the surface of the sample. No air plasma was observed in this condition. During the experiments, the sample was moved by a step motor in order to generate one plasma in...

Watch this Scientific Journal Video about Laser-induced Breakdown Spectroscopy: A New Approach for Nanoparticle's Mapping and Quantification in Organ Tissue at JoVE.com

Laser-induced Breakdown Spectroscopy: A New Approach for Nanoparticle&#039;s Mapping and Quantification in Organ Tissue

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.

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) ...

laser-induced-breakdown-spectroscopy-new-approach-for-nanoparticle-s

Research

JoVE Journal

Engineering

13.6K visualizações.  CNRS - Université Lyon 1.  Espectroscopia de decomposição induzida por laser realizada em órgão fina e tecido do tumor detectado com sucesso elementos naturais e gadolínio injetado artificialmente (Gd), emitidos a partir de nanopartículas à base de Gd. Imagens de elementos químicos atingiu uma resolução de 100 um e sensibilidade quantitativa sub-mM. A compatibilidade da instalação com microscopia óptica padrão enfatiza seu potencial para fornecer várias imagens de um mesmo tecido bi...