This work was partly supported by Grants-in-Aid for Scientific Research on Kiban-kenkyu A (No. 26249096), Innovative Areas &#34;Nano Informatics&#34; (No. 25106004), and Wakate-kenkyu B (No. 26870271) from the Japan Society of the Promotion of Science.

1. Sample preprocess
<ol>
	<li>Thin film preparation for TEM
	<ol>
		<li>Prepare a sample for the present analysis method using standard transmission electron microscopy (TEM) sample preparation techniques, such as electropolishing for metal materials, ion milling for semiconductors or ceramics, typically less than 100-200 nm for HARECXS, uniformly flat over the area of ~1 &#956;m. Prepare thinner (50-100 nm) samples for HARECES in general.</li>
	</ol>
	</li>
	<li>Sample mounting to TEM
	<ol>
		<li>Mount the thin film prepared on a double-tilting TEM sample holder, followed by inserting the holder into a TEM equipped with a scanning mode and an EDX detector (Figure 1).</li>
	</ol>
	</li>
</ol>
2. TEM operation (specific to JEM2100 STEM with beam-rocking option attached)
<ol>
	<li>TEM alignment for beam-rocking
	<ol>
		<li>Start the TEM operation. After the routine TEM beam alignment procedure, go to STEM mode by checking Attachment Scanning Image Display (ASID) in the ASID window in the TEM control monitor (TCM,&#160;Figure 1 &#38; Figure 2).</li>
	</ol>
	</li>
	<li>Optical axis alignment
	<ol>
		<li>Click the Rocking button in the ASID window of the TCM and then click the Spot button in the Simple Image Viewer (SIV) to stop beam-rocking (Figure 2). Remove the sample from the field of view. Set the beam-rocking range smaller than &#177;2&#176; by clicking the Mag increment/decrement buttons.</li>
		<li>Turn the Brightness knob on left operation panel (LOP: Figure 3) clockwise to the limit, followed by turning the OBJ FOCUS COARSE knob of Right Operation Panel (ROP: Figure 3) counterclockwise to an underfocused condition: a caustic spot (Figure 4) appears on the fluorescent viewing screen.</li>
		<li>Press the BRIGHT TILT function key (LOP) and move the caustic spot to the center of the fluorescent screen using a pair of DEF/STIG X/Y knobs (L/ROP).</li>
		<li>Press the Standard Focus button (ROP), and then turn the BRIGHTNESS knob back counterclockwise so that an alternative caustic spot appears on the fluorescent screen.</li>
		<li>Press the F3 function key (ROP) (or click the Spot button in &#39;Alignment Panel for Maintenance&#39; window on TCM) and move the beam spot to the center using a pair of DEF/STIG X/Y knobs.</li>
		<li>Repeat steps 2.2.2-2.2.5 until the beam position stays at center even if the lens condition is switched at step 2.2.2 and 2.2.4.</li>
	</ol>
	</li>
	<li>Incident beam collimation and setting its pivot point
	<ol>
		<li>Introduce the third largest condenser aperture at the center of the optical axis by turning the aperture knob clockwise with its position manually adjusted with two attached screws (Figure 1). Then, adjust the condenser lens stigmator to correct the beam shape to be coaxially defocused by turning the BRIGHTNESS knob both ways, using a pair of DEF/STIG knobs with COND STIG key on.</li>
		<li>Press the HT WOBB key (ROP) and adjust the BRIGHT TILT knob to minimize the beam size fluctuation with the change in acceleration voltage. This process adjusts the beam convergence angle minimum. Press the HT WOBB key again to stop HT wobbler.</li>
		<li>Activate maintenance mode (consult the manufacturer&#39;s manual). Select JEOL from the menu bar &#8594; Scan/Focus window&#160;&#8594; Scan Control tab in TCM. Then, click the Cor button and click the Scan button instead of Spot in the Image control panel of SIV.</li>
		<li>To minimize the beam shift with beam-rocking, adjust a pair of DEF/STIG knobs, followed by turning the OBJ FOCUS FINE knob slightly counterclockwise. Finally, match the sample and pivot point height using Z control keys (ROP) so that the sample is focused on the fluorescent screen.</li>
	</ol>
	</li>
	<li>Final beam alignment to obtain electron-channeling pattern of sample
	<ol>
		<li>Move the sample area of interest back to the center, and start beam-rocking by clicking the Scan button in the SIV window. Manually turn clockwise the annular dark field (ADF) detector cylinder (Figure 1) and insert the detector.</li>
		<li>Set the ADF detector position at the center of the beam position by adjusting a pair of DEF/STIG knobs with PLA key on (LOP: Figure 3). Check the STEI-DF button in the Image Select menu of the ASID window and the STEM monitor in the SIV window displays an electron-channeling pattern (ECP). Adjust the Brightness/Contrast in the ASID window to best see ECP. Slightly turn the BRIGHTNESS knob to see the ECP contrast sharpest.</li>
	</ol>
	</li>
	<li>Data acquisition for HARECXS by EDX
	<ol>
		<li>By operating the STEM in beam-rocking mode, collect the EDX spectra by following the conventional spectral image method (using the spectral imaging function in Figure 5) as a function of beam tilting angles in the x and y directions and display elemental intensity distribution for specified elements, as shown in Figure 5. 
		NOTE: The intensity distribution pattern is called an ionization channeling pattern (ICP).</li>
		<li>Use the Line Scan function in Figure 5 for the 1D tilting measurement of a systematic row of reflections. Yellow arrow appears in the ECP preview to specify the measuring range, as shown in the upper left panel in Figure 5. Stop measurements when sufficient data statistics are obtained for ICPs.</li>
	</ol>
	</li>
</ol>
3. Data analysis for quantification
<ol>
	<li>Express X-ray intensity Ix for impurity x in the following form as a function of X-ray intensity Ii of host element i,12&#160; 
	<img alt="Equation 1" src="/files/ftp_upload/62015/62015eq01v2.jpg" /> 
	where 
	<img alt="Equation 2" src="/files/ftp_upload/62015/62015eq02v2.jpg" /> 
	NOTE: Here, fix is the fractional occupancy of impurity x on the type i host site, cx is the concentration of impurity x, and ni is the fractional concentration of the type i host element&#160;among the total host sites prior to the accommodation of impurity atoms of type x. ki is the k-factor of the type i host element. The additional constant offset &#946;x has been introduced as an extra fitted parameter to account for differences in interaction delocalization and errors in background subtractions. &#945;ix can be derived from Eq. (1) by multivariate linear regression for many sampling points of the ICP X-ray intensities.</li>
	<li>Derive cx and fix utilizing the condition&#160;&#931;ifix&#160;= 1 as12 
	<img alt="Equation 3" src="/files/ftp_upload/62015/62015eq03v2.jpg" /> 
	The uncertainties in cx and fix for multiple impurities are readily derived from the error propagation principle: 
	<img alt="Equation 4" src="/files/ftp_upload/62015/62015eq04v2.jpg" /> 
	and 
	<img alt="Equation 5" src="/files/ftp_upload/62015/62015eq05v2.jpg" /> 
	where&#160;&#948; &#945;ix is the statistical error obtained in the linear regression from Eq. (1).&#160;</li>
</ol>

<ol>
	<li>Rietveld, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+profile+refinement+method+for+nuclear+and+magnetic+structures.">A profile refinement method for nuclear and magnetic structures.</a> Journal of Applied Crystallography. 2, 65-71 (1969).</li><li>Izumi, F., Ikeda, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Rietveld-analysis+program+RIETAN-98+and+its+applications+to+zeolites.">A Rietveld-analysis program RIETAN-98 and its applications to zeolites.</a> Materials Science Forum. 321-324, 198-203 (2000).</li><li>Rose, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optics+of+high-performance+electron+microscopes.">Optics of high-performance electron microscopes.</a> Science and Technology of Advanced Materials. 9, 014107 (2008).</li><li>Muller, D. 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=Atomic-scale+chemical+imaging+of+composition+and+bonding+by+aberration-+corrected+microscopy.">Atomic-scale chemical imaging of composition and bonding by aberration- corrected microscopy.</a> Science. 319, 1073-1076 (2008).</li><li>Spence, J. C. H., Taft&#248;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ALCHEMI:+A+new+technique+for+locating+atoms+in+small+crystals.">ALCHEMI: A new technique for locating atoms in small crystals.</a> Journal of Microscopy. 130, 147-154 (1982).</li><li>Taft&#248;, J., Spence, J. C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+site+location+of+iron+and+trace+elements+in+an+Mg-Fe+olivine+using+a+new+crystallographic+technique.">Crystal site location of iron and trace elements in an Mg-Fe olivine using a new crystallographic technique.</a> Science. 218, 49-51 (1982).</li><li>Yasuda, K., Yamamoto, T., Matsumura, 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+atomic+structure+of+disordered+ion+tracks+in+magnesium+aluminate+spinel.">The atomic structure of disordered ion tracks in magnesium aluminate spinel.</a> Journal of Microscopy. 59, 27 (2007).</li><li>Tatsumi, K., Muto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+electronic+structure+analysis+by+site-selective+ELNES+using+electron+channeling+and+first-principles+calculations.">Local electronic structure analysis by site-selective ELNES using electron channeling and first-principles calculations.</a> Journal of Physics Condensed Matter. 21, 1-14 (2009).</li><li>Yamamoto, Y., Tatsumi, K., Muto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-selective+electronic+structure+of+aluminum+in+oxide+ceramics+obtained+by+TEM-EELS+analysis+using+the+electron+standing-wave+method.">Site-selective electronic structure of aluminum in oxide ceramics obtained by TEM-EELS analysis using the electron standing-wave method.</a> Materials Transactions. 48, 2590-2594 (2007).</li><li>Tatsumi, K., Muto, S., Nishida, I., Rusz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+electronic+configurations+of+Fe+3d+states+by+energy+loss+by+channeled+electrons.">Site-specific electronic configurations of Fe 3d states by energy loss by channeled electrons.</a> Applied Physics Letters. 96, 201911 (2010).</li><li>Tatsumi, K., Muto, S., Rusz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+loss+by+channeled+electrons:+A+quantitative+study+on+transition+metal+oxides.">Energy loss by channeled electrons: A quantitative study on transition metal oxides.</a> Microscopy and Microanalysis. 19, 1586-1594 (2013).</li><li>Muto, S., Ohtsuka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-precision+quantitative+atomic-site-analysis+of+functional+dopants+in+crystalline+materials+by+electron-channelling-enhanced+microanalysis.">High-precision quantitative atomic-site-analysis of functional dopants in crystalline materials by electron-channelling-enhanced microanalysis.</a> Progress in Crystal Growth and Characterization of Materials. 63, 40-61 (2017).</li><li>Yamamoto, Y., 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=Quantitative+analysis+of+cation+mixing+and+local+valence+states+in+LiNixMn2-xO4+using+concurrent+HARECXS+and+HARECES+measurements.">Quantitative analysis of cation mixing and local valence states in LiNixMn2-xO4 using concurrent HARECXS and HARECES measurements.</a> Microscopy. 65, 253-262 (2016).</li><li>Ohtsuka, M., Muto, S., Tatsumi, K., Kobayashi, Y., Kawata, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+determination+of+occupation+sites+of+trace+Co+substituted+for+multiple+statistical+beam-rocking+TEM-EDXS+analysis.">Quantitative determination of occupation sites of trace Co substituted for multiple statistical beam-rocking TEM-EDXS analysis.</a> Microscopy. 65, 127-137 (2016).</li><li>Ohtsuka, M., Oda, K., Tanaka, M., Kitaoka, S., Muto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2D-HARECXS+analysis+of+dopant+and+oxygen+vacancy+sites+in+Al-doped+yttrium+titanate.">2D-HARECXS analysis of dopant and oxygen vacancy sites in Al-doped yttrium titanate.</a> J. Amer. Ceram. Soc. , (2021).</li><li>. QED for DigitalMicrograph Available from: <a target="_blank" href="https://www.hremresearch.com/Eng/plugin/QEDEng.html">https://www.hremresearch.com/Eng/plugin/QEDEng.html</a> (2020)</li></ol>

The authors have nothing to disclose.

Critical steps in the protocol are the ability to accurately align the incident rocking beam that has a small convergence angle with the pivot point, which is immobile at the specified area described in steps 2.2-2.3. A collimated incident beam with a convergence semi-angle of approximately no larger than 2 mrad was used. A beam size of 400 nm and diameter of 1 &#181;m can be selected by setting the condenser aperture #4 (10 &#956;m in diameter) and #3 (30 &#956;m) in the present hardware system.
The advantages of the present method are that (i) no advanced STEM instruments such as aberration-corrected STEM or even field emission electron gun is necessary; (ii) many sampling points (e.g., ~4,000 points for a scan area of 64 &#215; 64 pixels2) can be automatically collected with high efficiency, while operating the conventional STEM spectral imaging procedure on the analyzer side, and (iii) multiple spectroscopic methods such as EDX, EELS, and cathodoluminescence can be concurrently operated in a single integrated system, which enables multimodal analysis13.
Since the experimental ICPs can be precisely predicted by theoretical simulation, the method can be applied not only to cases where the crystal of interest contains multiple inequivalent atomic sites for a doped element14. Further extensions are ongoing, such as to detect the vacancy concentrations and associated displacements of host elements15, and even the ordering of dopants segregated along the grain boundaries of ceramics. The present method can provide a significant alternative technique applicable to relatively thick samples in contrast to atomic column-by-column analysis using aberration-corrected STEM, which requires the preparation of very thin high-quality samples (&#60; 10 nm).
Atom site-selective electronic state analysis using TEM-EELS (HARECES) rather than EDX is feasible8,9,10,11. For automatic measurement it is recommended to use &#39;ALCHEMI option&#39; in a beam controlling software &#39;QED,&#39; running on the Gatan Microscope Suite, supplied by HREM Research Inc16. In HARECES measurement, it is necessary to ensure that the transmitted beam is away from the EELS detector position and perpendicular to the systematic row in the beam tilting sequence8.
A limitation of this method is the minimum beam size of the incident electron beam, which limits the minimum measured area to approximately 400 nm. This is due to the aberration of the TEM lens system wherein the pivot center moves farther than the beam radius for a smaller beam size, which could be amended in the future by modifying the TEM deflector lens current setting to compensate for the beam wandering.
If the microscope used does not have beam-rocking mode, a very similar operation is achieved using QED software, which also addresses the limitation, as the software can rectify the pivot point moving even in the nano-beam mode. For S/TEMs manufactured by FEI Company (now part of Thermo Fisher Scientific), TIA scripting, open-source code can manage all S/TEM functions and attached detectors via a PC. Sequential EDX/EELS data acquisitions with successive incident beam tilting were performed using the scripting program TIA running on the TEM imaging and analysis platform13.

With the demand of downsizing most current industrial products, it is getting more and more important to understand the physical/chemical properties of materials from the microscopic perspective, sometimes in terms of atomic-scale spatial/electronic structures. Novel properties are often discovered unexpectedly when synthesizing materials by trial and error, selecting different numbers or kinds of elements, although current measurement techniques and ab initio theoretical calculations based on density functional theory have enabled design of novel materials with improved properties without time-consuming trial and error experiments. For example, some of the host atoms are substituted with other elements that can possibly improve the target property as results of either experimental or theoretical considerations. In this context, an important component of experimental information is brought about from detailed knowledge of the position of each constituent in the atomic structure of the material.
X-ray and/or neutron diffraction methods are conventionally and widely used not only because the structural analysis based on Rietveld analysis1,2 techniques has been well established and open to the public, but also owing to the development of high-flux X-ray sources (e.g., synchrotron radiation facilities) and modern neutron sources, which are easily accessible to general researchers. However, these techniques require samples with homogeneous structures, and they also require the Rietveld fit between the experimental and theoretical sets of diffracted peak intensities using structural factors. It can thus be difficult to distinguish between different elements if their structural factors are close to each other, such as in X-ray diffraction of neighboring elements in the periodic table.
In most current advanced materials, the compositions, precipitates, grain size, and impurities are adjusted and optimized to maximize the desired role at the nanometer scale. This means that these materials require characterization at the nanometer scale or even sub-nanometer scale to investigate whether they are synthesized as designed. In this context, it could be best achieved using transmission electron microscopy (TEM) and related analytical techniques.
The recent dramatic development of scanning TEM (STEM) in these decades, particularly based on aberration correction technologies, has accelerated a state-of-the-art technique to reveal the structure of a material and its elemental distribution at an atomic scale3,4. This method, however, requires precisely setting the crystalline material parallel to a low-order zone axis and to the extreme stability of the instrument during the measurement, which is a drawback. Hence, we demonstrate an alternative method that requires no such limitations, aberration&#160;correction, or even field emission electron gun.
Electron channeling in a crystalline material occurs if an incident electron beam propagates along particular atomic planes or columns, which depends on the direction of the incident high-energy electron beam with respect to the crystal axes, where an appropriate set of Bragg reflections and the excitation error of each reflection in a TEM are selected. The site-specific energy-dispersive X-ray (EDX or sometimes conventionally EDS) analysis technique that uses electron channeling is called the atom location by channeled electron microanalysis (ALCHEMI) method to evaluate the occupancies of host atomic sites by impurities5,6. This method has been extended to a more complex and quantitatively reliable approach, called high-angular-resolution electron-channeling X-ray spectroscopy (HARECXS), to determine impurity/dopant occupancies. This is realized by comparing the experimental beam-rocking curves with theoretical simulations7. This technique is further extended to high-angular-resolution electron-channeling electron spectroscopy (HARECES), which records electron energy loss spectra (EELS) instead of EDX8. This provides information on the site-specific local chemical states of a given element in different atomic environments9,10,11. In cases where each host element occupies a single crystallographic site, a simple linear regression and application of several formulae to the experimental dataset quantitatively determines the site occupancies of doped impurities without any theoretical simulations.
In the following sections, we provide detailed procedures of the method specific to the Jeol JEM2100 STEM system because it is explicitly equipped with the beam-rocking mode in the STEM operation menu. For users of other microscopes, please refer to the descriptions in the final paragraph of the Discussion section of this article.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Electron Energy-Loss Spectrometer</td>
			<td>Gatan Inc.</td>
			<td>Enfina1000</td>
			<td>Parallel EELS detector</td>
		</tr>
		<tr>
			<td>Energy dispersive X-ray detector</td>
			<td>JEOL Ltd.</td>
			<td>SD30GV</td>
			<td>EDS silicon drift detector</td>
		</tr>
		<tr>
			<td>Gatan Microscope Suite (GMS)</td>
			<td>Gatan Inc.</td>
			<td>ver. 2.3.</td>
			<td>Integrated software platform for controling cameras, detectors, S/TEM and data analysis</td>
		</tr>
		<tr>
			<td>QED</td>
			<td>HREM Research Inc.</td>
			<td>for GMS 2.3 32bit</td>
			<td>beam controlling software, running on the Gatan Microscope Suite</td>
		</tr>
		<tr>
			<td>scanning transmission electron microscope</td>
			<td>JEOL Ltd.</td>
			<td>JEM-2100</td>
			<td>Beam-rocking mode option in ASID controlling window</td>
		</tr>
		<tr>
			<td>TEMCON</td>
			<td>JEOL Ltd.</td>
			<td></td>
			<td>Control software for JEM 2100</td>
		</tr>
		<tr>
			<td>Thermo NSS software</td>
			<td>Thermo Fischer Scientific Inc., USA</td>
			<td></td>
			<td>EDS control software</td>
		</tr>
	</tbody>
</table>

quantitative atomic-site analysis of functional dopants/point defects in crystalline materials by electron-channeling-enhanced microanalysis

A novel elemental and chemical analysis scheme based on electron-channeling phenomena in crystalline materials is introduced, where the incident high-energy electron beam is rocked with the submicrometric pivot point fixed on a specimen. This method enables us to quantitatively derive the site occupancies and site-dependent chemical information of impurities or intentionally doped functional elements in a specimen, using energy-dispersive X-ray spectroscopy and electron energy-loss spectroscopy attached to a scanning transmission electron microscope, which is of significant interest to current materials science, particularly related to nanotechnologies. This scheme is applicable to any combination of elements even when the conventional Rietveld analysis by X-ray or neutron diffraction occasionally fails to provide the desired results because of limited sample sizes and close scattering factors of neighboring elements in the periodic table. In this methodological article, we demonstrate the basic experimental procedure and analysis method of the present beam-rocking microanalysis.

The experimental ECP for BaTiO3 and ICPs of Ba-L, Ti-K&#945;, and O-K&#945; near the [100] and [110] zone axes are shown in Figure 6A and Figure 6B, respectively. Each constituent element exhibits a specific ICP, indicating that the ICP is atomic site-specific12.
As a fundamental application example, we examined Eu3+-doped Ca2SnO4, which exhibits strong red emission derived from the 5D0-7F2 electric dipole transition of trivalent Eu ions (Eu3+). Considering the ionic radii similarity criterion, it would be more relevant to assume that Eu3+ occupies the Ca2+ sites because Eu3+ is significantly close in size to Ca2+ than to Sn4+. However, Rietveld analysis of powder X-ray diffraction data revealed that Eu3+ equally occupied the Ca2+ and Sn4+ sites, presumably because the local charge neutrality criterion dominates in this case. An Eu and Y co-doped sample Ca1.8Y0.2Eu0.2Sn0.8O4 was then synthesized because Y3+ ions with a smaller ionic radius preferentially occupy smaller cation (Sn4+) sites, expelling larger Eu3+ ions out of the Sn4+ site into the larger Ca2+ site without changing the charge balance. As expected, Ca1.8Y0.2Eu0.2Sn0.8O4 exhibited a stronger emission than the Ca1.9Eu0.2Sn0.9O4 sample. The stronger red emission in the co-doped sample is explained by the increased fraction of Eu3+ ions occupying the asymmetric Ca site, coordinated by seven oxygen atoms, which enhances the electric dipole moment compared to that of the symmetric six-coordinated Sn site.
A series of Eu and Y co-doped polycrystalline samples with nominal compositions of Ca1.9Eu0.2Sn0.9O4 and Ca1.8Eu0.2Y0.2Sn0.8O4 were prepared, and the site occupancies of the dopants were determined by the present method.
Figure 7&#160;shows the ECP and ICPs of Ca-K, Sn-L, O-K, Eu-L, and Y-L for the Ca1.8Eu0.2Y0.2Sn0.8O4 sample near the [100] zone. The Eu-L ICP was closer to the Ca-K ICP, whereas the Y-L ICP was closer to Sn-L ICP. This suggests that the Eu and Y occupation sites could be biased, as expected. The coefficients, &#945;ix for i = Ca, Sn, and x = Eu, Y derived using Eq. (1), where nCa = 2/3 and nSn = 1/3. The k-factors of the constituent elements are calibrated in advance using a reference material with a known composition, the detailed discussion of which is found in ref.12. The site occupancies fix (Eq. (3)) of the impurities, and the impurity concentrations c of all the samples are tabulated in Table 1.
In Ca1.9Eu0.2Sn0.9O4, Eu3+ occupied the Ca2+ and Sn4+ sites equally, consistent with the results of the XRD-Rietveld analysis. In contrast, Eu3+ and Y3+ occupied the Ca2+ and Sn4+ sites at ratios of approximately 7:3 and 4:6, respectively, in the co-doped samples, significantly biased as expected, but also maintaining the charge neutrality condition within the present experimental accuracies12.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62015/62015fig01.jpg" /> 
Figure 1: Instrumental outlook. Jeol JEM2100 STEM and its associated monitors, detectors, and operation panel configurations. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62015/62015fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62015/62015fig2v2.jpg" /> 
Figure 2: Layout of TEM control monitor (TCM). Control windows necessary for the present method are displayed and key functions and buttons are labeled. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62015/62015fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62015/62015fig3v2.jpg" /> 
Figure 3: Left/right operation panels of the S/TEM. (Left) Left operation panel (LOP). (Right) Right operation panel. The function keys and operation knobs necessary for the present method are labeled. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62015/62015fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62015/62015fig04.jpg" /> 
Figure 4: Caustic spot image on the fluorescent screen. The diameter of the spot ranges a few centimeters on the screen, depending on the defocus value. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62015/62015fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62015/62015fig05.jpg" /> 
Figure 5: Appearance of EDS&#160;control monitor. Electron-channeling pattern (ECP) preview in upper left panel specifies the area of measurement. For 1D tilting measurements, X-ray Linescan is selected in the leftmost panel and the range of measurement is indicated by the yellow arrow in the ECP preview. Periodic table in the lower left panel selects the elements of the ionization channeling patterns (ICPs) to be displayed in upper right panel. Lower right panel displays the accumulated EDS&#160;pattern in real-time. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62015/62015fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62015/62015fig06.jpg" /> 
Figure 6: Experimental ECPs and ICPs. (A: from left to right) ECP and ICPs of Ba-L, T-Ka, and O-Ka emissions from BaTiO3 obtained by beam-rocking near [100] zone axis. (B: from left to right) Same as (A) near [110] zone axes. This figure has been modified from [12]. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62015/62015fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62015/62015fig07.jpg" /> 
Figure 7. ECP and corresponding X-ray ICPs from Ca1.8Eu0.2Y0.2Sn0.8O4 by beam-rocking near the [100] zone axis. (A) ECP. (B-F) ICPs of Ca-Ka, Sn-L, O-Ka, O-Ka, Eu-L, and Y-L emissions, respectively. This figure has been modified from [12]. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62015/62015fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:font-size="7px" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample</td>
			<td>Dopant</td>
			<td>&#945;Ca</td>
			<td>&#945;Sn</td>
			<td>fCa</td>
			<td>fSn</td>
			<td>c&#160;x (x = Eu or Y)</td>
		</tr>
		<tr>
			<td>Ca1.9Eu0.2Sn0.9O4</td>
			<td>Eu</td>
			<td>1.71&#177;0.001</td>
			<td>0.083&#177;0.001</td>
			<td>0.57&#177;0.001</td>
			<td>0.43&#177;0.002</td>
			<td>0.061&#177;0.001</td>
		</tr>
		<tr>
			<td rowspan="2">Ca1.8Eu0.2Y0.2Sn0.8O4</td>
			<td>Eu</td>
			<td>0.162&#177;0.001</td>
			<td>0.077&#177;0.001</td>
			<td>0.78&#177;0.003</td>
			<td>0.22&#177;0.008</td>
			<td>0.088&#177;0.006</td>
		</tr>
		<tr>
			<td>Y</td>
			<td>0.040&#177;0.002</td>
			<td>0.265&#177;0.009</td>
			<td>0.28&#177;0.002</td>
			<td>0.72&#177;0.001</td>
			<td>0.118&#177;0.004</td>
		</tr>
	</tbody>
</table>
Table 1. Derived parameters (defined in text) of the samples of Ca2-xEuxSn1-yYyO4 where (x, y) = (0.2, 0.0) and (0.2, 0.2).

Watch this Scientific Journal Video about Quantitative Atomic-Site Analysis of Functional Dopants/Point Defects in Crystalline Materials by Electron-Channeling-Enhanced Microanalysis at JoVE.com

Quantitative Atomic-Site Analysis of Functional Dopants/Point Defects in Crystalline Materials by Electron-Channeling-Enhanced Microanalysis

We provide a general outline of quantitative microanalysis methods for estimating the site occupancies of impurities and their chemical states by taking advantage of electron-channeling phenomena under incident electron beam-rocking conditions, which reliably extract information from minority species, light elements, oxygen vacancies, and other point/line/planar defects.

A novel elemental and chemical analysis scheme based on electron-channeling phenomena in crystalline materials is introduced, where the incident ...

quantitative-atomic-site-analysis-functional-dopantspoint-defects

Nanyang Technological University

Research

JoVE Journal

Chemistry

5.5K Views.  Nagoya University. We provide a general outline of quantitative microanalysis methods for estimating the site occupancies of impurities and their chemical states by taking advantage of electron-channeling phenomena under incident electron beam-rocking conditions, which reliably extract information from minority species, light elements, oxygen vacancies, and other point/line/planar defects. 

Video: Quantitative Atomic-Site Analysis of Functional Dopants/Point Defects in Crystalline Materials by Electron-Channeling-Enhanced Microanalysis

Fabricating Nanogaps by Nanoskiving

There are several methods of fabricating nanogaps with controlled spacings, but the precise control over the sub-nanometer spacing between two electrodes-and generating them in practical quantities-is still challenging. The preparation of nanogap electrodes using nanoskiving, which is a form of edge lithography, is a fast, simple and powerful technique. This method is an entirely mechanical process which does not include any photo- or electron-beam lithographic steps and does not require any special equipment or infrastructure such as clean rooms. Nanoskiving is used to fabricate electrically addressable nanogaps with control over all three dimensions; the smallest dimension of these structures is defined by the thickness of the sacrificial layer (Al or Ag) or self-assembled monolayers. These wires can be manually positioned by transporting them on drops of water and are directly electrically-addressable; no further lithography is required to connect them to an electrometer.

The fabrication of electrically addressable, high-aspect-ratio (> 1000:1) metal nanowires separated by gaps of single nanometers using either sacrificial layers of aluminum and silver or self-assembled monolayers as templates is described. These nanogap structures are fabricated without a clean room or any photo- or electron-beam lithographic processes by a form of edge lithography known as nanoskiving.

There are several methods of fabricating nanogaps with controlled spacings, but the precise control over the sub-nanometer spacing between two ...

A Guided Materials Screening Approach for Developing Quantitative Sol-gel Derived Protein Microarrays

Microarrays have found use in the development of high-throughput assays for new materials and discovery of small-molecule drug leads. Herein we describe a guided material screening approach to identify sol-gel based materials that are suitable for producing three-dimensional protein microarrays. The approach first identifies materials that can be printed as microarrays, narrows down the number of materials by identifying those that are compatible with a given enzyme assay, and then hones in on optimal materials based on retention of maximum enzyme activity. This approach is applied to develop microarrays suitable for two different enzyme assays, one using acetylcholinesterase and the other using a set of four key kinases involved in cancer. In each case, it was possible to produce microarrays that could be used for quantitative small-molecule screening assays and production of dose-dependent inhibitor response curves. Importantly, the ability to screen many materials produced information on the types of materials that best suited both microarray production and retention of enzyme activity. The materials data provide insight into basic material requirements necessary for tailoring optimal, high-density sol-gel derived microarrays.

A guided material screening approach to develop sol-gel derived protein doped microarrays using an emerging pin-printing method of fabrication is described. This methodology is demonstrated through the development of acetylcholinesterase and multikinase microarrays, which are used for cost-effective small-molecule screening.

Microarrays have found use in the development of high-throughput assays for new materials and discovery of small-molecule drug leads. Herein we describe a ...

Layer-by-layer Synthesis and Transfer of Freestanding Conjugated Microporous Polymer Nanomembranes

CMP as large surface area materials have attracted growing interest recently, due to their high variability in the incorporation of functional groups in combination with their outstanding thermal and chemical stability, and low densities. However, their insoluble nature causes problems in their processing since usually applied techniques such as spin coating are not available. Especially for membrane applications, where the processing of CMP as thin films is desirable, the processing problems have hindered their commercial application.
Here we describe the interfacial synthesis of CMP thin films on functionalized substrates via molecular layer-by-layer (l-b-l) synthesis. This process allows the preparation of films with desired thickness and composition and even desired composition gradients.
The use of sacrificial supports allows the preparation of freestanding membranes by dissolution of the support after the synthesis. To handle such ultra-thin freestanding membranes the protection with sacrificial coatings showed great promise, to avoid rupture of the nanomembranes. To transfer the nanomembranes to the desired substrate, the coated membranes are upfloated at the air-liquid interface and then transferred via dip coating.

In this paper we describe the interfacial synthesis of conjugated microporous polymers (CMP) on sacrificial substrates, and the dissolution of the substrate for the preparation of freestanding CMP nanomembranes. In addition, we will describe how the fragile nanomembranes can be transferred to other substrates.

CMP as large surface area materials have attracted growing interest recently, due to their high variability in the incorporation of functional groups in ...

Determination of Carbonyl Functional Groups in Bio-oils by Potentiometric Titration: The Faix Method

Carbonyl compounds present in bio-oils are known to be responsible for bio-oil property changes upon storage and during upgrading. Specifically, carbonyls cause an increase in viscosity (often referred to as &#39;aging&#39;) during storage of bio-oils. As such, carbonyl content has previously been used as a method of tracking bio-oil aging and condensation reactions with less variability than viscosity measurements. Additionally, carbonyls are also responsible for coke formation in bio-oil upgrading processes. Given the importance of carbonyls in bio-oils, accurate analytical methods for their quantification are very important for the bio-oil community. Potentiometric titration methods based on carbonyl oximation have long been used for the determination of carbonyl content in pyrolysis bio-oils. Here, we present a modification of the traditional carbonyl oximation procedures that results in less reaction time, smaller sample size, higher precision, and more accurate carbonyl determinations. While traditional carbonyl oximation methods occur at room temperature, the Faix method presented here occurs at an elevated temperature of 80 &#176;C.

Here we present a potentiometric titration technique for accurately quantifying carbonyl compounds in pyrolysis bio-oils.

Carbonyl compounds present in bio-oils are known to be responsible for bio-oil property changes upon storage and during upgrading. Specifically, carbonyls ...

Controlled Photoredox Ring-Opening Polymerization of O-Carboxyanhydrides Mediated by Ni/Zn Complexes

Here, we describe an effective protocol that combines photoredox Ni/Ir catalysis with the use of a Zn-alkoxide for efficient ring-opening polymerization, allowing for the synthesis of isotactic poly(&#945;-hydroxy acids) with expected molecular weights (&#62;140 kDa) and narrow molecular weight distributions (Mw/Mn &#60; 1.1). This ring-opening polymerization is mediated by Ni and Zn complexes in the presence of an alcohol initiator and a photoredox Ir catalyst, irradiated by a blue LED (400 - 500 nm). The polymerization is performed at a low temperature (-15 &#176;C) to avoid undesired side reactions. The complete monomer consumption can be achieved within 4 - 8 hours, providing a polymer close to the expected molecular weight with narrow molecular weight distribution. The resulted number-average molecular weight shows a linear correlation with the degree of polymerization up to 1000. The homodecoupling 1H NMR study confirms that the obtained polymer is isotactic without epimerization. This polymerization reported herein offers a strategy for achieving rapid, controlled O-carboxyanhydrides polymerization to prepare stereoregular poly(&#945;-hydroxy acids) and its copolymers bearing various functional side-chain groups.

Controlled Photoredox Ring-Opening Polymerization of O-Carboxyanhydrides Mediated by Ni/Zn Complexes

A protocol for the controlled photoredox ring-opening polymerization of O-carboxyanhydrides mediated by Ni/Zn complexes is presented.

Here, we describe an effective protocol that combines photoredox Ni/Ir catalysis with the use of a Zn-alkoxide for efficient ring-opening polymerization, ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. These cell lines, after treatment with ascorbic acid (AA) and &#946;-glycerophosphate (&#946;-GP), undergo complete osteogenic transdifferentiation from proliferation to mineralization and produce matrix vesicles (MVs) that trigger apatite nucleation in the extracellular matrix (ECM).
Based on Alizarin Red-S (AR-S) staining and analysis of the composition of minerals in cell lysates using ultraviolet (UV) light or in vesicles using TEM imaging followed by EDX quantitation and ion mapping, we can infer that osteosarcoma Saos-2 and osteoblastic hFOB 1.19 cells reveal distinct mineralization profiles. Saos-2 cells mineralize more efficiently than hFOB 1.19 cells and produce larger mineral deposits that are not visible under UV light but are similar to hydroxyapatite (HA) in that they have more Ca and F substitutions.
The results obtained using these techniques allow us to conclude that the process of mineralization differs depending on the cell type. We propose that, at the cellular level, the origin and properties of vesicles predetermine the type of minerals.

We present a protocol to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. Their mineralization profiles were analyzed by Alizarin Red-S (AR-S) staining, ultraviolet (UV) light visualization, transmission electron microscopy (TEM) imaging and energy dispersive X-ray microanalysis (EDX).

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in ...

Chemical Synthesis of Porous Barium Titanate Thin Film and Thermal Stabilization of Ferroelectric Phase by Porosity-Induced Strain

Barium titanate (BaTiO3, hereafter BT) is an established ferroelectric material first discovered in the 1940s and still widely used because of its well-balanced ferroelectricity, piezoelectricity, and dielectric constant. In addition, BT does not contain any toxic elements. Therefore, it is considered to be an eco-friendly material, which has attracted considerable interest as a replacement for lead zirconate titanate (PZT). However, bulk BT loses its ferroelectricity at approximately 130 &#176;C, thus, it cannot be used at high temperatures. Because of the growing demand for high-temperature ferroelectric materials, it is important to enhance the thermal stability of ferroelectricity in BT. In previous studies, strain originating from the lattice mismatch at hetero-interfaces has been used. However, the sample preparation in this approach requires complicated and expensive physical processes, which are undesirable for practical applications.
In this study, we propose a chemical synthesis of a porous material as an alternative means of introducing strain. We synthesized a porous BT thin film using a surfactant-assisted sol-gel method, in which self-assembled amphipathic surfactant micelles were used as an organic template. Through a series of studies, we clarified that the introduction of pores had a similar effect on distorting the BT crystal lattice, to that of a hetero-interface, leading to the enhancement and stabilization of ferroelectricity. Owing to its simplicity and cost effectiveness, this fabrication process has considerable advantages over conventional methods.

Here, we present a protocol for the synthesis of porous barium titanate (BaTiO3) thin film by a surfactant-assisted sol-gel method, in which self-assembled amphipathic surfactant micelles are used as an organic template.

Barium titanate (BaTiO3, hereafter BT) is an established ferroelectric material first discovered in the 1940s and still widely used because of its ...

Microfluidic Preparation of Liquid Crystalline Elastomer Actuators

This paper focuses on the microfluidic process (and its parameters) to prepare actuating particles from liquid crystalline elastomers. The preparation usually consists in the formation of droplets containing low molar mass liquid crystals at elevated temperatures. Subsequently, these particle precursors are oriented in the flow field of the capillary and solidified by a crosslinking polymerization, which produces the final actuating particles. The optimization of the process is necessary to obtain the actuating particles and the proper variation of the process parameters (temperature and flow rate) and allows variations of size and shape (from oblate to strongly prolate morphologies) as well as the magnitude of actuation. In addition, it is possible to vary the type of actuation from elongation to contraction depending on the director profile induced to the droplets during the flow in the capillary, which again depends on the microfluidic process and its parameters. Furthermore, particles of more complex shapes, like core-shell structures or Janus particles, can be prepared by adjusting the setup. By the variation of the chemical structure and the mode of crosslinking (solidification) of the liquid crystalline elastomer, it is also possible to prepare actuating particles triggered by heat or UV-vis irradiation.

This article describes the microfluidic process and parameters to prepare actuating particles from liquid crystalline elastomers. This process allows the preparation of actuating particles and the variation of their size and shape (from oblate to strongly prolate, core-shell, and Janus morphologies) as well as the magnitude of actuation.

This paper focuses on the microfluidic process (and its parameters) to prepare actuating particles from liquid crystalline elastomers. The preparation ...

Exfoliation and Analysis of Large-area, Air-Sensitive Two-Dimensional Materials

We describe methods for producing and analyzing large, thin flakes of air-sensitive two-dimensional materials. Thin flakes of layered or van der Waals crystals are produced using mechanical exfoliation, in which layers are peeled off a bulk crystal using adhesive tape. This method produces high-quality flakes, but they are often small and can be hard to find, particularly for materials with relatively high cleavage energies such as black phosphorus. By heating the substrate and the tape, two-dimensional material adhesion to the substrate is promoted, and the flake yield can be increased by up to a factor of ten. After exfoliation, it is necessary to image or otherwise analyze these flakes but some two-dimensional materials are sensitive to oxygen or water and will degrade when exposed air. We have designed and tested a hermetic transfer cell to temporarily maintain the inert environment of a glovebox so that air-sensitive flakes can be imaged and analyzed with minimal degradation. The compact design of the transfer cell is such that optical analysis of sensitive materials can be performed outside of a glovebox without specialized equipment or modifications to existing equipment.

A method for exfoliating large thin flakes of air sensitive two-dimensional materials and safely transporting them for analysis outside of a glovebox is presented.

We describe methods for producing and analyzing large, thin flakes of air-sensitive two-dimensional materials. Thin flakes of layered or van der Waals ...