Name: obtaining 3d chemical maps by energy filtered transmission electron microscopy tomography
Uploaded: 2018-06-09T12:30:00
Description: Watch this Scientific Journal Video about Obtaining 3D Chemical Maps by Energy Filtered Transmission Electron Microscopy Tomography at JoVE.com

We are grateful to the French Ministry of Higher Education and Research, Conventions Industrielles de Formation par la Recherche (CIFRE) and IFP Energies Nouvelles for their financial support.

1. Sample Preparation
<ol>
	<li>Crush the sample in a mortar and disperse it in alcohol or distilled water; place a droplet of the sample on a microscopy grid and let it dry. 
	NOTE: Samples such as silica alumina or titania alumina can be a powder or an extruded material, and can be crushed and dispersed in a solution using ultrasound. In general, for ET analysis, it is important that the sample concentration on the grid is low, to avoid sample superposition and shadowing when tilting the grid at large angles. Th...

<ol>
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Mater. 8, 271-280 (2009).</li><li>Carenco, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+core+contribution+of+transmission+electron+microscopy+to+functional+nanomaterials+engineering.">The core contribution of transmission electron microscopy to functional nanomaterials engineering.</a> Nanoscale. 8 (3), 1260-1279 (2016).</li><li>Radon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uber+die+Bestimmung+von+Funktionen+durch+ihre+Integralwerte+langs+gewisser+Mannigfaltigkeiten.">Uber die Bestimmung von Funktionen durch ihre Integralwerte langs gewisser Mannigfaltigkeiten.</a> Akad. 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Today. 18 (7), 395-408 (2015).</li><li>Lepinay, K., Lorut, F., Pantel, R., Epicier, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+3D+tomography+of+28nm+high+K+metal+gate+transistor:+STEM+XEDS+experimental+method+and+results.">Chemical 3D tomography of 28nm high K metal gate transistor: STEM XEDS experimental method and results.</a> Micron. 47, 43-49 (2013).</li><li>Roiban, L., Sorbier, L., Pichon, C., Bayle-Guillemaud, P., Werckmann, J., Drillon, M., Ersen, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+Chemistry+of+Multiphase+Nanomaterials+by+Energy-Filtered+Transmission+Electron+Microscopy+Tomography.">Three-Dimensional Chemistry of Multiphase Nanomaterials by Energy-Filtered Transmission Electron Microscopy Tomography.</a> Microsc. Microanal. 18 (05), 1118-1128 (2012).</li><li>Roiban, L., Sorbier, L., Hirlimann, C., Ersen, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3+D+Chemical+Distribution+of+Titania-Alumina+Catalyst+Supports+Prepared+by+the+Swing-pH+Method.">3 D Chemical Distribution of Titania-Alumina Catalyst Supports Prepared by the Swing-pH Method.</a> ChemCatChem. 8 (9), 1651-1657 (2016).</li><li>Egerton, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Electron Energy-Loss Spectroscopy in the Electron Microscope. , (2011).</li><li>Messaoudi, C., Aschman, N., Cunha, M., Oikawa, T., Sorzano, C. O. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+visualization+of+three-dimensional+image+data+using+IMOD.">Computer visualization of three-dimensional image data using IMOD.</a> J Struct Biol. 116 (1), 71-76 (1996).</li><li>Align RGB planes. ImageJ Available from: <a target="_blank" href="https://ImageJ.net/Align_RGB_planes">https://ImageJ.net/Align_RGB_planes</a> (2018)</li><li>MessaoudiI, C., Boudier, T., Sorzano, C., Marco, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TomoJ:+tomography+software+for+three-dimensional+reconstruction+in+transmission+electron+microscopy.">TomoJ: tomography software for three-dimensional reconstruction in transmission electron microscopy.</a> BMC Bioinf. 8 (1), 288 (2007).</li><li>Saxton, W. 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The aim of this paper is to describe how to obtain 3D chemical maps using EFTEM tomography. This protocol is completely original and was developed by the authors.
EFTEM tomography as described here has several drawbacks: (i) Only samples that are electron beam resistant can be analyzed, due to the long exposure time needed for obtaining filtered images. (ii) EFTEM tomography is sensitive to the diffraction contrast. (iii) Many of the alignments were performed manually. To obtain the 3D chemica...

The properties of functional materials are dependent on their 3D parameters. To fully comprehend their properties and enhance their functions, it is important to analyze their morphology and chemical distribution in 3D. Electron tomography1 (ET) is one of the best techniques to provide this information at the nanometer scale2,3. It consists of rotating the sample over a large angular range and recording one image at each angular step. The obtained tilt series is used to reconstruct the volume of the sample by using mathematical algorithms based on the Radon transform4,5. Selecting grey levels in the volume helps to model the sample in 3D and quantify 3D parameters like particle localization6 and size distribution7, pore position and size distribution8, etc.
In general, ET is performed with an electron microscope by tilting the sample to the maximum possible angle, preferably more than 70&#176; in either direction. At each tilt angle, a projection of the sample is recorded forming an images tilt series. That tilt series is aligned and used to reconstruct the volume of the sample which will be segmented and quantified. Because the sample cannot be rotated from -90&#176; to +90&#176;, the reconstructed volume has an anisotropic resolution along the orthogonal axis9 due to the blind recording angle.
ET can be performed in different imaging modes. The bright field TEM mode (BF-TEM) is used to study amorphous materials, biological samples, polymers, or catalyst supports with complex shapes. The image analysis is based on the differentiation of the gray levels characterizing the density of the components10 (a dense component will be more dark than a lighter, i.e., less dense component). High-angle annular dark field in scanning TEM mode (HAADF-STEM) is used to analyze crystalline samples. The signal provides chemical information as a function of the atomic number; a heavy component of the sample will appear brighter that a lighter one9. Other modes, like Energy Dispersive X-ray spectroscopy (EDX), which collects the X-ray emitted by the material11, and energy filtered imaging mode (EFTEM)12,13, are also capable of assessing the 3D chemical distribution within the sample.
In EFTEM imaging, the 2D chemical maps can be recorded using a TEM with an electron energy spectrometer. The spectrometer acts as a magnetic prism by dispersing the electrons as a function of their energy. An image is created by the electrons depending on the energy lost from interacting with a specific atom. If the same 2D chemical map is computed at different tilt angles, a tilt series of chemical projections is obtained, which can be used to reconstruct the 3D chemical volume.
Not all the materials can be analyzed by EFTEM tomography. The technique is reserved for samples with weak or disordered materials. Nevertheless, it can be used for analyzing light elements that are very difficult to differentiate when using other imaging techniques. In addition, to obtain reliable 2D chemical maps, the thickness of the material is required to be less than the mean free path of the electrons through the material14. Under this condition, the probability of having a single electron interacting with a single atom is greatest. Two methods are used to calculate a 2D chemical map. The first one, and the most used is the &#34;three-windows method&#34;, where two filtered energy windows are recorded before the ionization edge of the element under analysis, and a third after the ionization edge13. The first two images are used to estimate the background, which is extrapolated using a power law at the position of the third window and subtracted from it. The obtained image is the projection of the 3D distribution of the analyzed chemical element in the sample volume. The second method is called the &#34;jump-ratio&#34;; it uses only two energy-filtered images, one before and one after the ionization edge. This method is qualitative, as the final image is calculated only by performing the ratio between those two images, and does not account for background energy variation.
By combining EFTEM with ET, the analytical tomography of the filtered energy can be obtained. EFTEM tomography and atom probe tomography (APT) are complementary techniques. As compared to APT, EFTEM tomography is a non-destructive characterization analysis that does not need complex sample preparation. It can be used to perform various characterizations on a unique nanoparticle. EFTEM tomography can analyze insulating materials, while APT needs at the very least laser assistance to measure them. APT runs at the atomic scale, while EFTEM tomography performs adequately with a lower resolution. EFTEM tomography is pertinent only for samples that resist beam degradation during the experiment. To record all the filtered images at all the tilted angles, the sample can be exposed to the electron beam for as long as 2 h. Moreover, to record a maximum chemical signal in the 2D maps, longer exposition durations at high beam intensity may be necessary. In such conditions, the beam sensitive samples suffer drastic morphological and chemical changes. Therefore, a precise measurement of the sample sensitivity to the electron beam must be established before the experiment. In addition, EFTEM tomography is the result of recording as many tomograms as necessary to determine the spatial location and nature of the chemical elements that are present in the sample. Nevertheless, EFTEM tomography can provide important information concerning the 3D chemical distribution for samples, such as catalyst supports, to give new insights for modeling their catalytic applications.
Today it is possible to use dedicated software that can select the energy interval, record filtered energy window images, and calculate the chemical maps at different tilt angles. They allow tilting the sample, tracking, focusing, and recording the filtered image in EFTEM mode. The 2D chemical maps can be calculated, and then the tilt series can be aligned, the chemical volume computed using iterative algorithms, and finally the series can be segmented and quantified15,16.

<table>
	<tbody>
		<tr>
			<td>JEOL 2100f</td>
			<td>JEOL</td>
			<td></td>
			<td>Electron microscope</td>
		</tr>
		<tr>
			<td>Tridiem Gatan Imaging Filter (GIF)</td>
			<td>Gatan</td>
			<td></td>
			<td>Post colum energy filter</td>
		</tr>
		<tr>
			<td>Digital micrograph</td>
			<td>Gatan</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Gatan EFTEM tomography plugin</td>
			<td>Gatan</td>
			<td></td>
			<td>Dedicated software to record filtered tilt series for EFTEM tomograohy</td>
		</tr>
		<tr>
			<td>Tomoj</td>
			<td>Imagej plugin http://www.cmib.fr/en/download/softwares/</td>
			<td></td>
			<td>Free software developed by Currie Institute in Paris, France for electron tomography</td>
		</tr>
		<tr>
			<td>EFTEM-Tomoj</td>
			<td>Imagej plugin http://www.cmib.fr/en/download/softwares/</td>
			<td></td>
			<td>Free software developed by Currie Institute in Paris, France , for EFTEM imaging</td>
		</tr>
		<tr>
			<td>Imod</td>
			<td>http://bio3d.colorado.edu/imod/</td>
			<td></td>
			<td>Free software developed by University of Colorado, USA for electron tomography</td>
		</tr>
		<tr>
			<td>Imagej</td>
			<td>https://imagej.nih.gov/ij/</td>
			<td></td>
			<td>Free software developed by National Institute of Mental Health, Bethesda, Maryland, USA for images treatment</td>
		</tr>
		<tr>
			<td>Merge channels</td>
			<td>https://imagej.net/Color_Image_Processing</td>
			<td></td>
			<td>Fonction in Imagej allowing to give different colors to volumes while they are overlapped</td>
		</tr>
		<tr>
			<td>3D Slicer</td>
			<td>https://www.slicer.org/</td>
			<td></td>
			<td>Free software developed by a large consortium lead by Ron Kikinis , Harvard Medical School, Boston, MA, SUA</td>
		</tr>
		<tr>
			<td>Chimera</td>
			<td>https://www.cgl.ucsf.edu/chimera/</td>
			<td></td>
			<td>Free software developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco,for data segmentation, cuatification and visualisation of 3D models</td>
		</tr>
		<tr>
			<td>silica alumina support of catalyst</td>
			<td>IFPEN</td>
			<td></td>
			<td>sample prepared for eleboration of this protocol</td>
		</tr>
		<tr>
			<td>titania alumina support of catalyst</td>
			<td>IFPEN</td>
			<td></td>
			<td>sample prepared for eleboration of this protocol</td>
		</tr>
		<tr>
			<td>alcohol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Au nanoparticles of 5 nm</td>
			<td>BBI Solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Holey carbn film 200 mesh microscopy grid</td>
			<td>Agar</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EDX sepctrometer</td>
			<td>Oxford Instruments</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

obtaining 3d chemical maps by energy filtered transmission electron microscopy tomography

Energy filtered transmission electron microscopy tomography (EFTEM tomography) can provide three-dimensional (3D) chemical maps of materials at a nanometric scale. EFTEM tomography can separate chemical elements that are very difficult to distinguish using other imaging techniques. The experimental protocol described here shows how to create 3D chemical maps to understand the chemical distribution and morphology of a material. Sample preparation steps for data segmentation are presented. This protocol permits the 3D distribution analysis of chemical elements in a nanometric sample. However, it should be noted that currently, the 3D chemical maps can only be generated for samples that are not beam sensitive, since the recording of filtered images requires long exposure times to an intense electron beam. The protocol was applied to quantify the chemical distribution of the components of two different heterogeneous catalyst supports. In the first study, the chemical distribution of aluminum and titanium in titania-alumina supports was analyzed. The samples were prepared using the swing-pH method. In the second, the chemical distribution of aluminum and silicon in silica-alumina supports that were prepared using the sol-powder and mechanical mixture methods was examined.

An example of the application of this protocol is shown in reference13. EFTEM tomography was used for analyzing titania alumina catalyst supports. To enhance the catalytic activity of the active phase of MoS2 nanoparticles, in applications like hydrodesulfurization (HDS), it is important that titania is preponderant at the support surface, and in contact with the active phase. It is known that titania has a smaller specific surface than alumina. The aim ...

Watch this Scientific Journal Video about Obtaining 3D Chemical Maps by Energy Filtered Transmission Electron Microscopy Tomography at JoVE.com

Obtaining 3D Chemical Maps by Energy Filtered Transmission Electron Microscopy Tomography

This paper describes a protocol to achieve 3D chemical maps combining energy filtered imaging and electron tomography. The chemical distribution of two catalyst supports formed by elements that are difficult to distinguish by other imaging techniques was studied. Each application consists of mapping overlapped chemical elements - respectively spaced-ionization edges.

Energy filtered transmission electron microscopy tomography (EFTEM tomography) can provide three-dimensional (3D) chemical maps of materials at a ...

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