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 1.325 M,&#160;CaCl2 20 mM to 100 &#181;l of H2O and 80 &#181;l of the primary solution of Gd-based nanoparticles to obtain a 200 &#181;l solution at 40 mM ready-to-inject (City: Villeurbanne, at lab).</li>
	<li>Inject the 200 &#181;l Gd-based nanoparticles solution intravenously&#160;into anesthetized tumor-bearing rodents (City: Lyon Sud (Oullins), 15 km from the lab).</li>
	<li>1-24 hr after injection, sacrifice the mice and put the biological samples into isopentane cooled by liquid nitrogen. Store the samples at - 80 &#176;C (City: Lyon Sud (Oullins), 15 km from the lab).</li>
	<li>Slice the sample (City: usually Grenoble, 100 km from the lab; I will try to have an access in Lyon Sud) in 100 &#181;m-thick slides and put the biological slides on specific plastic dishes (Petri dish). Store at -80 &#176;C. 
	Note: Plastic dishes are basically a very pure polymer sample. They are used to avoid interference with elements contained in the tissue.</li>
</ol>
2. Sample Preparation for Calibration
<ol>
	<li>Prepare vials with increasing doses of Gd-based nanoparticle into water (0 nM, 100 nM, 500 nM, 1 &#181;M, 5 &#181;M, 10 &#181;M, 50 &#181;M, 100 &#181;M, 500 &#181;M, 1 mM, and 5 mM).</li>
	<li>Put a 5 &#181;l-drop of each solution regularly spaced by 3 mm on the Petri dish.</li>
	<li>Dry at room temperature for 20 min.</li>
</ol>
3. LIBS Experiment
<ol>
	<li>Initializing the LIBS Setup
	<ol>
		<li>Laser Settings. After switching on the instruments, wait&#160;10-20 min for laser pulse energy stabilization and cooling down of the ICCD camera to -20 &#176;C. Adjust the pulse energy with the attenuator. 
		Note: The optimum laser parameters for mapping tissues are 5 nsec pulse duration, 1,064 nm wavelength, and pulse energy of about 4 mJ. The laser uses is a typical Nd:YAG nanosecond laser.</li>
		<li>LIBS Settings. Set the laser focusing position (with respect to the sample surface) to obtain the smaller crater diameter (about 50 &#181;m or less). 
		Note: This corresponds to a laser pulse focalization 100 &#181;m below the sample surface.</li>
		<li>Spectrometer Settings. Use the Czerny-Turner spectrometer combined with a 1,200 lines/mm grating and a high temporal resolution ICCD camera. Control all these devices by computer. Set the input slit value to 40 &#181;m. Set the spectral range regarding the element to be analyzed. Use the spectral range covering 325 to 355 nm to detect Gd with high sensitivity, as well as Na, Cu and Ca. Set the ICCD parameters with a delay of 300 nsec, a gate of 2 &#181;sec, and a gain of 200.</li>
	</ol>
	</li>
	<li>Mapping Measurement
	<ol>
		<li>Place the biological sample on the LIBS motorized sample holder.</li>
		<li>Adjust the height of the sample according to the laser focus position.</li>
		<li>Take a high resolution photograph of the sample slice.</li>
		<li>Set the mapping module of the LIBS acquisition software to perform a map with typically 100 x 100 measurement points spaced by a resolution of about 100 &#181;m.</li>
		<li>Start the acquisition. From this point automate everything; the moving sequence as well as the spectrum recording and saving. 40 min are required for a mapping of 10,000 points (equivalent to 1 cm2&#160;for 100 &#181;m resolution). Regroup all the recorded spectra into a same file.</li>
		<li>When finished, take a second photograph of the sample slice</li>
	</ol>
	</li>
	<li>Calibration Measurement</li>
</ol>
With the same experimental parameters, measure the calibration samples (for preparation details, see section 2). Perform a map or record 25 spectra (obtained from measurement sites in the center part of the drop) in each of the calibration drops.
4. LIBS Spectrum Analysis: Constructing of Chemical Images
<ol>
	<li>Record all LIBS spectra from the tissue mapping and load them in the LIBS software analysis. Subtract the baseline for each spectrum and construct the chemical images with relative intensity scale using a false color. 
	Note: An algorithm retrieves specific line intensities, such as Gd, Cu, Na, or Ca.</li>
	<li>Perform the same operation on the spectra measured from the calibrated sample to allow the calibration curves calculation (relation between intensity and concentration) and build a quantitative map or image for Gd (or other element of interest).</li>
	<li>Apply the adequate treatments to the chemical images, such as interpolation or smoothing. Save the intensity or concentration maps in image format (bitmap).</li>
</ol>

<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. 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The authors have nothing to disclose.

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) ...

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13.6K Aufrufe.  CNRS - Université Lyon 1.  Laser-Emissionsspektroskopie an dünnen Organ-und Tumorgewebe durchgeführt erfolgreich erkannt natürlichen Elemente und künstlich injiziert Gadolinium (Gd), von Gd-Nanopartikel auf der Basis ausgestellt. Bilder von chemischen Elementen erreicht eine Auflösung von 100 um und quantitative Unter mM Empfindlichkeit. Die Kompatibilität der Einrichtung mit optischen Standardmikroskop hebt das Potential, um mehrere Bilder von einer gleichen biologischen Gewebes bereitzuste...

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Angle-resolved Photoemission Spectroscopy At Ultra-low Temperatures

The physical properties of a material are defined by its electronic structure. Electrons in solids are characterized by energy (&omega;) and momentum (k) and the probability to find them in a particular state with given &omega; and k is described by the spectral function A(k, &omega;). This function can be directly measured in an experiment based on the well-known photoelectric effect, for the explanation of which Albert Einstein received the Nobel Prize back in 1921. In the photoelectric effect the light shone on a surface ejects electrons from the material. According to Einstein, energy conservation allows one to determine the energy of an electron inside the sample, provided the energy of the light photon and kinetic energy of the outgoing photoelectron are known. Momentum conservation makes it also possible to estimate k relating it to the momentum of the photoelectron by measuring the angle at which the photoelectron left the surface. The modern version of this technique is called Angle-Resolved Photoemission Spectroscopy (ARPES) and exploits both conservation laws in order to determine the electronic structure, i.e. energy and momentum of electrons inside the solid. In order to resolve the details crucial for understanding the topical problems of condensed matter physics, three quantities need to be minimized: uncertainty* in photon energy, uncertainty in kinetic energy of photoelectrons and temperature of the sample.
In our approach we combine three recent achievements in the field of synchrotron radiation, surface science and cryogenics. We use synchrotron radiation with tunable photon energy contributing an uncertainty of the order of 1 meV, an electron energy analyzer which detects the kinetic energies with a precision of the order of 1 meV and a He3 cryostat which allows us to keep the temperature of the sample below 1 K. We discuss the exemplary results obtained on single crystals of Sr2RuO4 and some other materials. The electronic structure of this material can be determined with an unprecedented clarity.

The overall goal of this method is to determine the low-energy electronic structure of solids at ultra-low temperatures using Angle-Resolved Photoemission Spectroscopy with synchrotron radiation.

The physical properties of a material are defined by its  electronic structure. Electrons in solids are characterized by energy (&omega;)  and momentum ...

Probing and Mapping Electrode Surfaces in Solid Oxide Fuel Cells

Solid oxide fuel cells (SOFCs) are potentially the most efficient and cost-effective solution to utilization of a wide variety of fuels beyond hydrogen 1-7. The performance of SOFCs and the rates of many chemical and energy transformation processes in energy storage and conversion devices in general are limited primarily by charge and mass transfer along electrode surfaces and across interfaces. Unfortunately, the mechanistic understanding of these processes is still lacking, due largely to the difficulty of characterizing these processes under in situ conditions. This knowledge gap is a chief obstacle to SOFC commercialization. The development of tools for probing and mapping surface chemistries relevant to electrode reactions is vital to unraveling the mechanisms of surface processes and to achieving rational design of new electrode materials for more efficient energy storage and conversion2. Among the relatively few in situ surface analysis methods, Raman spectroscopy can be performed even with high temperatures and harsh atmospheres, making it ideal for characterizing chemical processes relevant to SOFC anode performance and degradation8-12. It can also be used alongside electrochemical measurements, potentially allowing direct correlation of electrochemistry to surface chemistry in an operating cell. Proper in situ Raman mapping measurements would be useful for pin-pointing important anode reaction mechanisms because of its sensitivity to the relevant species, including anode performance degradation through carbon deposition8, 10, 13, 14 ("coking") and sulfur poisoning11, 15 and the manner in which surface modifications stave off this degradation16. The current work demonstrates significant progress towards this capability. In addition, the family of scanning probe microscopy (SPM) techniques provides a special approach to interrogate the electrode surface with nanoscale resolution. Besides the surface topography that is routinely collected by AFM and STM, other properties such as local electronic states, ion diffusion coefficient and surface potential can also be investigated17-22. In this work, electrochemical measurements, Raman spectroscopy, and SPM were used in conjunction with a novel test electrode platform that consists of a Ni mesh electrode embedded in an yttria-stabilized zirconia (YSZ) electrolyte. Cell performance testing and impedance spectroscopy under fuel containing H2S was characterized, and Raman mapping was used to further elucidate the nature of sulfur poisoning. In situ Raman monitoring was used to investigate coking behavior. Finally, atomic force microscopy (AFM) and electrostatic force microscopy (EFM) were used to further visualize carbon deposition on the nanoscale. From this research, we desire to produce a more complete picture of the SOFC anode.

We present a unique platform for characterizing electrode surfaces in solid oxide fuel cells (SOFCs) that allows simultaneous performance of multiple characterization techniques (e.g. in situ Raman spectroscopy and scanning probe microscopy alongside electrochemical measurements). Complementary information from these analyses may help to advance toward a more profound understanding of electrode reaction and degradation mechanisms, providing insights into rational design of better materials for SOFCs.

Solid oxide fuel cells (SOFCs) are potentially the most efficient and cost-effective solution to utilization of a wide variety of fuels beyond hydrogen ...

Scanning-probe Single-electron Capacitance Spectroscopy

The integration of low-temperature scanning-probe techniques and single-electron capacitance spectroscopy represents a powerful tool to study the electronic quantum structure of small systems - including individual atomic dopants in semiconductors. Here we present a capacitance-based method, known as Subsurface Charge Accumulation (SCA) imaging, which is capable of resolving single-electron charging while achieving sufficient spatial resolution to image individual atomic dopants. The use of a capacitance technique enables observation of subsurface features, such as dopants buried many nanometers beneath the surface of a semiconductor material1,2,3. In principle, this technique can be applied to any system to resolve electron motion below an insulating surface.
As in other electric-field-sensitive scanned-probe techniques4, the lateral spatial resolution of the measurement depends in part on the radius of curvature of the probe tip. Using tips with a small radius of curvature can enable spatial resolution of a few tens of nanometers. This fine spatial resolution allows investigations of small numbers (down to one) of subsurface dopants1,2. The charge resolution depends greatly on the sensitivity of the charge detection circuitry; using high electron mobility transistors (HEMT) in such circuits at cryogenic temperatures enables a sensitivity of approximately 0.01 electrons/Hz&frac12; at 0.3 K 5.

Scanning-probe single-electron capacitance spectroscopy facilitates the study of single-electron motion in localized subsurface regions. A sensitive charge-detection circuit is incorporated into a cryogenic scanning probe microscope to investigate small systems of dopant atoms beneath the surface of semiconductor samples.

The integration of low-temperature scanning-probe techniques and single-electron capacitance spectroscopy represents a powerful tool to study the ...

Measurement and Analysis of Atomic Hydrogen and Diatomic Molecular AlO, C2, CN, and TiO Spectra Following Laser-induced Optical Breakdown

In this work, we present time-resolved measurements of atomic and diatomic spectra following laser-induced optical breakdown. A typical LIBS arrangement is used. Here we operate a Nd:YAG laser at a frequency of 10 Hz at the fundamental wavelength of 1,064 nm. The 14 nsec pulses with anenergy of 190 mJ/pulse are focused to a 50 &#181;m spot size to generate a plasma from optical breakdown or laser ablation in air. The microplasma is imaged onto the entrance slit of a 0.6 m spectrometer, and spectra are recorded using an 1,800 grooves/mm grating an intensified linear diode array and optical multichannel analyzer (OMA) or an ICCD. Of interest are Stark-broadened atomic lines of the hydrogen Balmer series to infer electron density. We also elaborate on temperature measurements from diatomic emission spectra of aluminum monoxide (AlO), carbon (C2), cyanogen (CN), and titanium monoxide (TiO).
The experimental procedures include wavelength and sensitivity calibrations. Analysis of the recorded molecular spectra is accomplished by the fitting of data with tabulated line strengths. Furthermore, Monte-Carlo type simulations are performed to estimate the error margins. Time-resolved measurements are essential for the transient plasma commonly encountered in LIBS.

Measurement and Analysis of Atomic Hydrogen and Diatomic Molecular AlO, C2, CN, and TiO Spectra Following Laser-induced Optical Breakdown

Time-resolved atomic and diatomic molecular species are measured using LIBS. The spectra are collected at various time delays following the generation of optical breakdown plasma with Nd:YAG laser radiation&#160;and are analyzed to infer electron density and temperature.

In this work, we present time-resolved measurements of atomic and diatomic spectra following laser-induced optical breakdown. A typical LIBS arrangement ...

Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy

Monitoring the dynamics of protonation and protein backbone conformation changes during the function of a protein is an essential step towards understanding its mechanism. Protonation and conformational changes affect the vibration pattern of amino acid side chains and of the peptide bond, respectively, both of which can be probed by infrared (IR) difference spectroscopy. For proteins whose function can be repetitively and reproducibly triggered by light, it is possible to obtain infrared difference spectra with (sub)microsecond resolution over a broad spectral range using the step-scan Fourier transform infrared technique. With ~102-103 repetitions of the photoreaction, the minimum number to complete a scan at reasonable spectral resolution and bandwidth, the noise level in the absorption difference spectra can be as low as ~10-4, sufficient to follow the kinetics of protonation changes from a single amino acid. Lower noise levels can be accomplished by more data averaging and/or mathematical processing. The amount of protein required for optimal results is between 5-100 &#181;g, depending on the sampling technique used. Regarding additional requirements, the protein needs to be first concentrated in a low ionic strength buffer and then dried to form a film. The protein film is hydrated prior to the experiment, either with little droplets of water or under controlled atmospheric humidity. The attained hydration level (g of water / g of protein) is gauged from an IR absorption spectrum. To showcase the technique, we studied the photocycle of the light-driven proton-pump bacteriorhodopsin in its native purple membrane environment, and of the light-gated ion channel channelrhodopsin-2 solubilized in detergent.

Key steps of protein function, in particular backbone conformational changes and proton transfer reactions, often take place in the microsecond to millisecond time scale. These dynamical processes can be studied by time-resolved step-scan Fourier-transform infrared spectroscopy, in particular for proteins whose function is triggered by light.

Monitoring the dynamics of protonation and protein backbone conformation changes during the function of a protein is an essential step towards ...

In Situ Time-dependent Dielectric Breakdown in the Transmission Electron Microscope: A Possibility to Understand the Failure Mechanism in Microelectronic Devices

The time-dependent dielectric breakdown (TDDB) in on-chip interconnect stacks is one of the most critical failure mechanisms for microelectronic devices. The aggressive scaling of feature sizes, both on devices and interconnects, leads to serious challenges to ensure the required product reliability. Standard reliability tests and post-mortem failure analysis provide only limited information about the physics of failure mechanisms and degradation kinetics. Therefore it is necessary to develop new experimental approaches and procedures to study the TDDB failure mechanisms and degradation kinetics in particular. In this paper, an in situ experimental methodology in the transmission electron microscope (TEM) is demonstrated to investigate the TDDB degradation and failure mechanisms in Cu/ULK interconnect stacks. High quality imaging and chemical analysis are used to study the kinetic process. The in situ electrical test is integrated into the TEM to provide an elevated electrical field to the dielectrics. Electron tomography is utilized to characterize the directed Cu diffusion in the insulating dielectrics. This experimental procedure opens a possibility to study the failure mechanism in interconnect stacks of microelectronic products, and it could also be extended to other structures in active devices.

In Situ Time-dependent Dielectric Breakdown in the Transmission Electron Microscope: A Possibility to Understand the Failure Mechanism in Microelectronic Devices

The time-dependent dielectric breakdown (TDDB) in on-chip interconnect stacks is one of the most critical failure mechanisms for microelectronic devices. This paper demonstrates the procedure of an in situ TDDB experiment in the transmission electron microscope, which opens a possibility to study the failure mechanism in microelectronic products.

The time-dependent dielectric breakdown (TDDB) in on-chip interconnect stacks is one of the most critical failure mechanisms for microelectronic devices. ...

Laser-induced Forward Transfer for Flip-chip Packaging of Single Dies

Flip-chip (FC) packaging is a key technology for realizing high performance, ultra-miniaturized and high-density circuits in the micro-electronics industry. In this technique the chip and/or the substrate is bumped and the two are bonded via these conductive bumps. Many bumping techniques have been developed and intensively investigated since the introduction of the FC technology in 19601 such as stencil printing, stud bumping, evaporation and electroless/electroplating2. Despite the progress that these methods have made they all suffer from one or more than one drawbacks that need to be addressed such as cost, complex processing steps, high processing temperatures, manufacturing time and most importantly the lack of flexibility. In this paper, we demonstrate a simple and cost-effective laser-based bump forming technique known as Laser-induced Forward Transfer (LIFT)3. Using the LIFT technique a wide range of bump materials can be printed in a single-step with great flexibility, high speed and accuracy at RT. In addition, LIFT enables the bumping and bonding down to chip-scale, which is critical for fabricating ultra-miniature circuitry.

We demonstrate the use of the Laser-induced Forward Transfer (LIFT) technique for flip-chip assembly of optoelectronic components. This approach provides a simple, cost-effective, low-temperature, fast and flexible solution for fine-pitch bumping and bonding on chip-scale for achieving high-density circuits for optoelectronic applications.

Flip-chip (FC) packaging is a key technology for realizing high performance, ultra-miniaturized and high-density circuits in the micro-electronics ...

Energy Dispersive X-ray Tomography for 3D Elemental Mapping of Individual Nanoparticles

Energy dispersive X-ray spectroscopy within the scanning transmission electron microscope (STEM) provides accurate elemental analysis with high spatial resolution, and is even capable of providing atomically resolved elemental maps. In this technique, a highly focused electron beam is incident upon a thin sample and the energy of emitted X-rays is measured in order to determine the atomic species of material within the beam path. This elementally sensitive spectroscopy technique can be extended to three dimensional tomographic imaging by acquiring multiple spectrum images with the sample tilted along an axis perpendicular to the electron beam direction.
Elemental distributions within single nanoparticles are often important for determining their optical, catalytic and magnetic properties. Techniques such as X-ray tomography and slice and view energy dispersive X-ray mapping in the scanning electron microscope provide elementally sensitive three dimensional imaging but are typically limited to spatial resolutions of &gt; 20 nm. Atom probe tomography provides near atomic resolution but preparing nanoparticle samples for atom probe analysis is often challenging. Thus, elementally sensitive techniques applied within the scanning transmission electron microscope are uniquely placed to study elemental distributions within nanoparticles of dimensions 10-100 nm.
Here, energy dispersive X-ray (EDX) spectroscopy within the STEM is applied to investigate the distribution of elements in single AgAu nanoparticles. The surface segregation of both Ag and Au, at different nanoparticle compositions, has been observed.

The use of energy dispersive X-ray tomography in the scanning transmission electron microscope to characterize elemental distributions within single nanoparticles in three dimensions is described.

Energy dispersive X-ray spectroscopy within the scanning transmission electron microscope (STEM) provides accurate elemental analysis with high spatial ...

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 ...

Blast Quantification Using Hopkinson Pressure Bars

Near-field blast load measurement presents an issue to many sensor types as they must endure very aggressive environments and be able to measure pressures up to many hundreds of megapascals. In this respect the simplicity of the Hopkinson pressure bar has a major advantage in that while the measurement end of the Hopkinson bar can endure and be exposed to harsh conditions, the strain gauge mounted to the bar can be affixed some distance away. This allows protective housings to be utilized which protect the strain gauge but do not interfere with the measurement acquisition. The use of an array of pressure bars allows the pressure-time histories at discrete known points to be measured. This article also describes the interpolation routine used to derive pressure-time histories at un-instrumented locations on the plane of interest. Currently the technique has been used to measure loading from high explosives in free air and buried shallowly in various soils.

This protocol details the use of Hopkinson pressure bars to measure reflected blast loading from near-field explosive events. It is capable of interpolating a pressure-time history at any point on a reflective boundary and as such can be used to fully characterize the spatial and temporal variations in loading produced.

Near-field blast load measurement presents an issue to many sensor types as they must endure very aggressive environments and be able to measure pressures ...