The authors kindly acknowledge financial support from the &#34;Akademische Gesellschaft Lippstadt&#34; as well as from the &#34;Ministerium f&#252;r Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen&#34;. Photographs in Figures 1, 2 and 5 courtesy to Markus Horstmann, Hamm-Lippstadt University of Applied Sciences.

1. Sample Preparation for X-ray Computed Tomography (CT)
<ol>
	<li>Glue sample (LED cf. materials section) to a 2 mm &#216; hollow carbon fiber bar of appropriate length using hot-melt adhesive.</li>
	<li>Adjust position of specimen by using a hot-air gun if needed. Fix the sample in the CT-sample chamber using the three-jaw chuck.</li>
</ol>
2. CT Measurement Setup
<ol>
	<li>Perform warmup and centering procedures according to the control software of the X-ray tube. 
	NOTE: Use tube-control software of manufacturers&#39; CT and standard protocol as specified by the supplier (cf. materials section).</li>
	<li>Calibrate beam and detector using data acquisition software, such as that specified in the materials section. Determine dark current and adjust offset and gain of the detector according to the standard procedures provided by the manufacturer (cf. materials section).</li>
	<li>Adjust imaging parameters. For the results shown here, use the following adjustments: Set image magnification to 36.37, set Voxel Size to 1.37 &#181;m, set number of images to 1,800 (per 360&#176;), set imaging time to 500 msec, set number of averaged images to 3 and number of skipped frames to 1, set image size to 2,284 x 2,304 pixel.</li>
	<li>Adjust measurement parameters. For the results shown use the following adjustments: Set focus object distance (FOD) to 5.5 mm, set focus detector distance (FDD) to 200 mm, set X-ray tube voltage to 100 kV and tube current to 135 &#181;A, use 0.2 mm Cu foil for beam hardening.</li>
</ol>
3. Performance of CT Scan
NOTE: X-ray intensities may vary during the measurement. To compensate for these eventual fluctuations, a region of interest (ROI) window is placed where the X-rays will not interfere with the sample. This region is not affected by X-ray absorption through the sample, it is therefore the region with the highest measured intensity.
<ol>
	<li>Select the ROI by identifying the area not obscured by the measured object during one full rotation. In the measurement window with the live image, press and hold the left mouse button and draw up a red framed window.</li>
	<li>Right click on this window&#39;s frame to open a context menu. Then select &#34;set as observation window&#34;. The frame&#39;s color will change to yellow, and the observation window will be fixed in the measurement window. 
	NOTE: Using this software function therefore sets the observation window and defines the region in the scanned-images, where the X-rays do not interact with the sample. This is to correct for possible drift of gray-values for rays, which directly hit the detector (free rays, causing the gray-value of air). It is the brightest region in the image during a full rotation of the sample. 
	NOTE: Due to the fact that heating of the X-ray tube will lead to thermal expansions of tube materials, a software module is activated which corrects for such effects. These effects cause shifts in the X-ray foci on the target as well as spatially, which during the measurement will cause a movement of the measured object in the recorded images.
	<ol>
		<li>Activate the software module &#34;auto scan optimizer&#34;, through which nine images are taken before the actual scan of the sample. These images are taken in 40&#176; steps, while rotating the sample. 
		NOTE: This software module will in addition to the correction of thermal effects also allow for the correction of smaller mechanical movements of the sample itself. The module is found in the graphical user interface of the measurement software.</li>
		<li>Additionally activate the module &#34;detector shift routine&#34;. The simultaneous activation of these two modules before starting the actual CT scan ensures correction for movements of the sample and for ring artifacts. 
		NOTE: This software module is used to reduce ring artifacts: The detector is moved to a position ca. &#177;10 pixels from the initial position and all images taken are averaged. This reduces the influence faulty pixels.</li>
		<li>Use the &#34;auto scan optimizer&#34; and &#34;detector shift routine&#34; of the acquisition software for the purpose described above, the two modules are selected separately and are used simultaneously in this investigation.</li>
	</ol>
	</li>
	<li>Scan sample by starting the &#34;data acquisition routine&#34; in the acquisition software.</li>
</ol>
4. Reconstruction of Volume Information, Planning of Micro Preparation
<ol>
	<li>Use manufacturers&#39; reconstruction software to render volume information. Volume rendering is carried out digitally using a computing cluster to reconstruct the sample features presented by X-ray absorption.</li>
	<li>Apply image correction algorithms: bhc+ (beam hardening correction) applying the value for &#34;different materials&#34; (which is 5.8) to remove beam hardening and scan optimizer to remove unwanted sample movements cf. 3.2). Carry these steps out according to the supplier&#39;s software manual (cf. materials section).</li>
	<li>Choose an area for reconstruction, and define a region of interest (ROI). In this case the ROI is defined by the volume the LED occupies during a full circle described by its rotation in the CT sample chamber. Make use of the software options &#34;use observation&#34; and &#34;ROI CT-filter&#34; to suppress artifacts, stick to the supplier&#39;s software manual (cf. materials section), when doing this.</li>
	<li>Reconstruct the volume for ROI. After setting ROI, filters and correction options in the reconstruction software, carry out the volume reconstruction using the computing cluster as specified by the instrument&#39;s supplier (cf. materials section).</li>
	<li>Transfer reconstruction data to the CT-data-analysis software, align sample in xy, xz and yz planes using the &#34;simple registration&#34; function in the software. Apply &#34;median&#34; filtering, using filter size &#34;3&#34;. 
	NOTE: Carry the following steps out as described in the software manual (cf. materials section).
	<ol>
		<li>Using the software, inspect the rendered volume, and check for the electrical interconnects in the device structure to ensure supply of electrical current from the soldering pads underneath the device to the light emitting semiconductor chip on top.</li>
		<li>Define cutting position and amount of sample to be removed by grinding and polishing for the subsequent micro preparation, such that after removal the device is still operational (avoid open circuitry). Use distance and measurement tools of the software to ensure operability of the specimen after micro preparation (length may be calibrated by the known LED-chip dimensions of 1 mm x 1 mm).</li>
	</ol>
	</li>
</ol>
5. Micro Preparation
<ol>
	<li>Solder silver wire to the anode and cathode pads of the LED manually. Use solder wire of 1 mm diameter and with the composition 60% Sn, 39% Pb and 1% Cu. Ensure appropriate positioning of the wires.</li>
	<li>Embed LED in epoxy-resin using transparent supports (e.g., rings of 25 mm or 40 mm diameter). Drill two small holes on opposite sides of the support and feed the silver wire (which contacts the LED) through it. Position LED by means of tightening or loosening the silver wire to align the front edge of the LED and the support.
	<ol>
		<li>Fill the ring with epoxy inside a silicone beaker pretreated to ensure that it will not stick to the epoxy and subsequently let the epoxy harden.</li>
	</ol>
	</li>
	<li>Using a stereomicroscope, visually ensure that support and LED are aligned. Mechanically remove any resin, which is in excess (e.g., outside of the support), by grinding with coarse abrasive paper.</li>
	<li>Fix the LED (embedded in the epoxy resin), in a planar fashion to a sample holder , for precision grinding.</li>
	<li>Use a grinder with abrasion measurement and remove the sample surface up to 100 &#181;m from the targeted plane&#39;s position.</li>
	<li>Carefully remove further material on a manually operated grinder using 9 &#181;m diamond suspension. Control the progress in abrasion frequently with a stereo microscope.</li>
	<li>On reaching the targeted region, as defined by the CT scan, switch to 3 &#181;m diamond suspension and finally suitable polishing suspensions, by changing to the corresponding grinding and polishing discs of the manual grinder used. Control the progress in short intervals with a stereo microscope. 
	NOTE: Ideally the prepared surface will now correspond to the target plane defined in the CT measurement.</li>
	<li>In steps 5.5 and 5.6 always remove grinding and polishing suspensions before using the microscope by rinsing with de-ionized water and wiping with cotton pads.</li>
	<li>After polishing, observe the smooth and scratch free surface using a stereo microscope. Clean the specimen with de-ionized water and cotton pads, and remove water by rinsing with ethanol (pure methylated spirit) and drying using a hair dryer.</li>
	<li>Check the specimen for electrical operability, i.e., current flow through the light emitting diode in forward direction and no current flow in reverse direction, using a digital multimeter.</li>
</ol>
6. LM Measurement Setup
<ol>
	<li>Mount specimen in appropriate sample holder for CLEM (cf. materials section). Ensure that the sample holder fixes the sample for use in LM, Sputter Coater and SEM.</li>
	<li>Adjust calibration marks (L-structures on the holder) to the same height as sample surface (ca. 4 mm). Ensure that the polished surface is parallel to focal plane of the LM. Fix sample holder onto the motorized xy-stage of LM. Connect LED to power supply. The power supply should operate in constant current mode.</li>
	<li>Calibrate sample holder position on the xy-stage by saving the position of calibration marks as reference points. 
	NOTE: A detailed instruction for this step including semi-automatic procedure is described in the user manual (cf. materials section).</li>
</ol>
7. LM Characterization
<ol>
	<li>Move xy-stage of LM such that ROI of the sample is in the field of view of the LM. Ensure that the LM camera has an accurate white balance by auto-calibration as provided in the LM-software and use of a white reference surface (e.g., sheet of paper).</li>
	<li>Perform LM imaging in a compound LM with reflected light according to the steps described in the user manual provided by the supplier (cf. materials section). For the results shown here the bright field, dark field, and polarization contrast were imaged with a 50X objective.</li>
	<li>Switch on the power supply and tune LED emission. Switch off LM illumination and adjust exposure time of the LM camera (ca. 92 msec dependent on emission intensity). Obtain LM image of light distribution within the sample (Luminescence contrast).</li>
	<li>If applicable, image luminescence together with other contrasts by activating LM illumination and LED simultaneously. 
	NOTE: Otherwise, images with different contrasts can also be mixed by means of image processing later on.</li>
	<li>Save all LM images together with the corresponding stage position as described in the user manual provided by the supplier (cf. materials section).</li>
</ol>
8. Sputter Coating
<ol>
	<li>Remove sample holder from LM and power supply. Make sure that the sample remains stably fixed within the holder.</li>
	<li>Fix Copper conductive tape onto the polished sample surface around the LED and contacting the sample holder. Do not cover the ROIs with the tape.</li>
	<li>Using a foil cover the sample holder and prepare a window similar to the sample diameter (ca. 5 mm). Fix the complete sample holder within the foil such that the window is directly above the sample.</li>
	<li>Place the sample holder into the recipient of the sputter coater ensuring that the sample surface can be coated. Sputter a 5 nm thick carbon layer onto the sample surface (from carbon rod). Move the sample holder out of the sputter coater and remove the foil.</li>
</ol>
9. SEM Measurement Setup
<ol>
	<li>Mount sample holder onto the SEM adapter and place it on the motorized stage of SEM. Pump the vacuum chamber.</li>
	<li>Calibrate sample holder position within the SEM by saving the position of calibration marks as reference points. 
	NOTE: A detailed instruction for this step including semi-automatic procedure is described in the user manual (cf. materials section).</li>
	<li>Define coordinate transformation from LM to SEM stage for direct relocation of ROIs and for navigation within the LM images. This step can also be done automatically by software as described in the user manual (cf. materials section).</li>
</ol>
10. SEM Analysis
<ol>
	<li>Move stage to show ROI on sample and perform SEM analysis in the same location as in the LM.</li>
	<li>Select &#34;SE detection&#34; for surface imaging. Choose electron energy of 20 keV, set the aperture to 30 &#181;m and position the sample at a working distance of 8.7 mm.</li>
	<li>Select &#34;BSE detection&#34; for material contrast. Choose electron energy of 20 keV, set the aperture to 30 &#181;m and position the sample at a working distance of 8.7 mm.</li>
	<li>Select &#34;EDX detection&#34; for element mapping. Choose electron energy of 20 keV, set the aperture to 60 &#181;m and position the sample at a working distance of 9 mm. Detect the following elements: Y, Al, Ca, Si, Ga, Au, Ni, and Cu.</li>
</ol>
11. Image Processing
<ol>
	<li>Perform overlay of LM and SEM images by selecting identical points in the images from LM and SEM and by further image processing as described in the user manual provided by the supplier (cf. materials section).</li>
</ol>

<ol>
	<li>Mueller-Mach, R., Mueller, G. O., Krames, M. R., Trottier, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-power+phosphor-converted+light-emitting+diodes+based+on+III-Nitrides.">High-power phosphor-converted light-emitting diodes based on III-Nitrides.</a> IEEE J. Sel. Top. Quantum Electron. 8 (2), 339-345 (2002).</li><li>Branas, C., Azcondo, F. J., Alonso, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid-State+Lighting:+A+System+Review.">Solid-State Lighting: A System Review.</a> IEEE Ind. Electron. Mag. 7 (4), 6-14 (2013).</li><li>Chang, M. -. H., Das, D., Varde, P. V., Pecht, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+emitting+diodes+reliability+review.">Light emitting diodes reliability review.</a> Microelectron. Reliab. 52 (5), 762-782 (2012).</li><li>Ayodha, T., Han, H. S., Kim, J., Kim, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+chip+die+bonding+on+thermal+resistance+of+high+power+LEDs.">Effect of chip die bonding on thermal resistance of high power LEDs.</a> Intersoc. Conf. Therm. Thermomechanical Phenom. Electron. Syst. ITHERM. , 957-961 (2012).</li><li>Cason, M., Estrada, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+X-ray+MicroCT+for+non-destructive+failure+analysis+and+package+construction+characterization.">Application of X-ray MicroCT for non-destructive failure analysis and package construction characterization.</a> Proc. Int. Symp. Phys. Fail. Anal. Integr. Circuits, IPFA. , (2011).</li><li>Chen, R., Zhang, Q., Peng, T., Jiao, F., Liu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Failure+analysis+techniques+for+high+power+light+emitting+diodes.">Failure analysis techniques for high power light emitting diodes.</a> 2011 12th Int. Conf. Electron. Packag. Technol. High Density Packag. , 1-4 (2011).</li><li>Chen, Z., Zhang, Q., 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=Study+on+the+reliability+of+application-specific+led+package+by+thermal+shock+testing,+failure+analysis,+and+fluid-solid+coupling+thermo-mechanical+simulation.">Study on the reliability of application-specific led package by thermal shock testing, failure analysis, and fluid-solid coupling thermo-mechanical simulation.</a> IEEE Trans. Components, Packag. Manuf. Technol. 2 (7), 1135-1142 (2012).</li><li>Luniak, M., Holtge, H., Brodmann, R., Wolter, K. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Characterization+of+Electronic+Packages+with+Confocal+Microscopy.">Optical Characterization of Electronic Packages with Confocal Microscopy.</a> 2006 1st Electron. Syst. Technol. Conf. 2 (16), 1813-1815 (2006).</li><li>Marks, M. R., Hassan, Z., Cheong, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+Methods+for+Ultrathin+Wafer+and+Die+Quality:+A+Review.">Characterization Methods for Ultrathin Wafer and Die Quality: A Review.</a> IEEE Trans. Components, Packag. Manuf. Technol. 4 (12), 2042-2057 (2014).</li><li>Rosc, J., Hammer, 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=Reliability+assessment+of+contact+wires+in+LED-devices+using+in+situ+X-ray+computed+tomography+and+thermo-mechanical+simulations.">Reliability assessment of contact wires in LED-devices using in situ X-ray computed tomography and thermo-mechanical simulations.</a> Proc. 5th Electron. Syst. Technol. Conf. , 1-6 (2014).</li><li>Zhaohui, C., Qin, Z., Kai, W., Xiaobing, L., Sheng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+test+and+failure+analysis+of+high+power+LED+packages.">Reliability test and failure analysis of high power LED packages.</a> J. Semicond. 32 (1), 014007 (2011).</li><li>Hamon, B., Bataillou, B., Hamon, B., Mendizabal, L., Gasse, A., Feuillet, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-contacts+degradation+analysis+of+white+flip+chip+LEDs+during+reliability+tests.">N-contacts degradation analysis of white flip chip LEDs during reliability tests.</a> 2014 IEEE Int. Reliab. Phys. Symp. , FA.1.1-FA.1.6 (2014).</li><li>Tsai, M. -. Y., Tang, C. -. Y., Yen, C. -. Y., Chang, L. -. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bump+and+Underfill+Effects+on+Thermal+Behaviors+of+Flip-Chip+LED+Packages:+Measurement+and+Modeling.">Bump and Underfill Effects on Thermal Behaviors of Flip-Chip LED Packages: Measurement and Modeling.</a> IEEE Trans. Device Mater. Reliab. 14 (1), 161-168 (2014).</li><li>Wang, F. -. K., Lu, Y. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Useful+lifetime+analysis+for+high-power+white+LEDs.">Useful lifetime analysis for high-power white LEDs.</a> Microelectron. Reliab. 54 (6-7), 1307-1315 (2014).</li><li>Liu, Y., Zhao, J., Yuan, C. C. -. A., Zhang, G. Q., Sun, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chip-on-Flexible+Packaging+for+High-Power+Flip-Chip+Light-Emitting+Diode+by+AuSn+and+SAC+Soldering.">Chip-on-Flexible Packaging for High-Power Flip-Chip Light-Emitting Diode by AuSn and SAC Soldering.</a> IEEE Trans. Components, Packag. Manuf. Technol. 4 (11), 1754-1759 (2014).</li><li>Thomas, C., Edelmann, M., Lysenkov, D., Hafner, C., Bernthaler, T., Schneider, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlative+Light+and+Electron+Microscopy+(CLEM)+for+Characterization+of+Lithium+Ion+Battery+Materials.">Correlative Light and Electron Microscopy (CLEM) for Characterization of Lithium Ion Battery Materials.</a> Microsc. Microanal. 16, 784-785 (2010).</li><li>Thomas, C., Ogbazghi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlative+Microscopy+of+Optical+Materials.">Correlative Microscopy of Optical Materials.</a> Imaging &amp; Microscopy. 3, 32-34 (2014).</li></ol>

The authors have nothing to disclose.

The advantages of this multimodal approach consist in the location-dependent correlation of the acquired data. The multimodal approach described here should be contrasted in subsequent analyses with each technique separately. For example, luminescence properties visible in LM can be linked to compositions as detected using SEM/EDS. The volume information obtained by CT can be extended with in depth analyses of cross-sections prepared in a targeted fashion. CT data also enable fast location of possible areas of interest i...

This article demonstrates the potential and advantages of a combination of X-ray computed tomography (CT) with correlative light and electron microscopy (CLEM) for the exemplary in depth characterization of light emitting diodes (LED). With this technique it is possible to plan the micro preparation of the LED in such a fashion that while a cross section can be imaged microscopically the electrical functionality is preserved in the remainder of the specimen. The procedure has several unique features: firstly, the planned micro preparation by aid of the rendered volume of the entire sample obtained by CT; secondly, the observation of the LED by light microscopy (LM) with the complete variety of imaging techniques available (bright and dark field, polarization contrast, etc.); thirdly, observation of the LED in operation by LM; fourthly, observation of identical regions with the full variety of electron microscopy imaging techniques comprising secondary electron (SE) and back scatter electron (BSE) imaging, as well as energy dispersive X-ray fluorescence spectroscopy (EDX).
LEDs for illumination applications are designed to emit white light, although in certain applications color variability may be favorable. This broad emission cannot be achieved by emission from one compound semiconductor, since LEDs emit radiation in a narrow spectral band (circa 30 nm full width half maximum (FWHM)). Therefore white LED light is commonly generated by the combination a blue LED with phosphors which convert the short-wavelength radiation into broad emission over a large spectral range1. Color variable LED solutions usually make use of at least three primaries, which generally results in higher market prices.2
The use of either CT, LM or SEM is of course well established (e.g., in failure analysis for LEDs3-15), however the comprehensive and purposeful combination of all three techniques described here may offer new insights and will enable faster tracks towards meaningful characterization results.
From 3D microstructural analysis of the packaged device in CT the regions of interest (ROIs) can be identified and selected. With this non-destructive method, electrical connections can also be identified and considered for further preparation. The precise preparation of a 2D cross section allows investigations of the device in operation despite the destructive nature of this method. The cross section can now be characterized by CLEM16,17 which enables a very efficient and flexible characterization of identical ROIs with LM as well as SEM. By this approach, the advantages of both microscopy techniques can be combined. For example, a fast identification of ROIs in the LM is followed by high-resolution imaging in the SEM. But furthermore, the correlation of information from the LM (e.g., color, optical properties, particle distribution) with the visualization and analysis techniques of the SEM (e.g., particle size, surface morphology, element distribution) allows a deeper understanding of functional behavior and microstructure within a white LED.

<table><tbody><tr><td>X-ray Computer Tomograph</td><td>General Electric</td><td>not applicable</td><td>type: nanotom s research edition</td></tr><tr><td>acquisition software</td><td>General Electric</td><td>not applicable</td><td>phoenix Datos| x2 acquisition and corresponding manual</td></tr><tr><td>reconstruction software</td><td>General Electric</td><td>not applicable</td><td>phoenix Datos| x2 acquisition and corresponding manual</td></tr><tr><td>rendering software</td><td>Volume Graphics</td><td>not applicable</td><td>VGStudio Max 2.2 and corresponding manual</td></tr><tr><td>grinder (manual)</td><td>Struers</td><td>5296327</td><td>Labopol 21</td></tr><tr><td>sample holder</td><td>Struers</td><td>4886102</td><td>UniForce</td></tr><tr><td>grinder (automated)</td><td>Struers</td><td>6026127</td><td>Tegramin 25</td></tr><tr><td>epoxy resin/hardener</td><td>Struers</td><td>40200030/40200031</td><td>Epoxy fix resin / Epoxy fix hardener</td></tr><tr><td>Ethanol</td><td>Struers</td><td>950301</td><td>Kleenol</td></tr><tr><td>Light Microscope</td><td>Zeiss</td><td>not applicable</td><td>Axio Imager M2m&amp;nbsp;</td></tr><tr><td>Electron Microscope</td><td>Zeiss</td><td>not applicable</td><td>Sigma&amp;nbsp;</td></tr><tr><td>CLEM software</td><td>Zeiss</td><td>not applicable</td><td>Axio Vision SE64 Rel.4.9 and corresponding manual</td></tr><tr><td>CLEM sample holder</td><td>Zeiss</td><td>432335-9101-000</td><td>Specimen holder CorrMic MAT Universal B</td></tr><tr><td>SEM Adapter for CLEM sample holder</td><td>Zeiss</td><td>432335-9151-000</td><td>SEM Adapter for Specimen holder CorrMic MAT Universal B</td></tr><tr><td>sputter coater</td><td>Quorum</td><td>not applicable</td><td>Q150TES</td></tr><tr><td>EDS detector</td><td>R&amp;ouml;ntec</td><td>not applicable</td><td>X-Flash 1106</td></tr><tr><td>solder</td><td>Stannol</td><td>535251</td><td>type: HS10</td></tr><tr><td>LED</td><td>Lumileds</td><td>not applicable</td><td>LUXEON Rebel warm white, research sample</td></tr></tbody></table>

in depth analyses of leds by a combination of x-ray computed tomography (ct) and light microscopy (lm) correlated with scanning electron microscopy (sem)

In failure analysis, device characterization and reverse engineering of light emitting diodes (LEDs), and similar electronic components of micro-characterization, plays an important role. Commonly, different techniques like X-ray computed tomography (CT), light microscopy (LM) and scanning electron microscopy (SEM) are used separately. Similarly, the results have to be treated for each technique independently. Here a comprehensive study is shown which demonstrates the potentials leveraged by linking CT, LM and SEM. In depth characterization is performed on a white emitting LED, which can be operated throughout all characterization steps. Major advantages are: planned preparation of defined cross sections, correlation of optical properties to structural and compositional information, as well as reliable identification of different functional regions. This results from the breadth of information available from identical regions of interest (ROIs): polarization contrast, bright and dark-field LM images, as well as optical images of the LED cross section in operation. This is supplemented by SEM imaging techniques and micro-analysis using energy dispersive X-ray spectroscopy.

The characterized LED is shown in Figure 1. It is a white emitting LED with a Chip size of 1 x 1 mm2 and a partially ceramic luminescent color converter. Gluing the LED in a slightly slanted position onto a carbon fiber bar avoids CT artifacts caused by sample symmetry (Figure 2). Results of the CT measurement allow for planning the position of the cross section of the sample, and ensure electrical operability after partial abrasion (Fi...

Watch this Scientific Journal Video about In Depth Analyses of LEDs by a Combination of X-ray Computed Tomography CT and Light Microscopy LM Correlated with Scanning Electron Microscopy SEM at JoVE.co

In Depth Analyses of LEDs by a Combination of X-ray Computed Tomography CT and Light Microscopy LM Correlated with Scanning Electron Microscopy SEM

A workflow for comprehensive micro-characterization of active optical devices is outlined. It contains structural as well as functional investigations by means of CT, LM and SEM. The method is demonstrated for a white LED which can be still be operated during characterization.

In Depth Analyses of LEDs by a Combination of X-ray Computed Tomography (CT) and Light Microscopy (LM) Correlated with Scanning Electron Microscopy (SEM)

In failure analysis, device characterization and reverse engineering of light emitting diodes (LEDs), and similar electronic components of ...

in-depth-analyses-leds-combination-x-ray-computed-tomography-ct-light

Universidad de Cartagena UDEC

Research

JoVE Journal

Engineering

9.2K Views.  Hamm-Lippstadt University of Applied Sciences. A workflow for comprehensive micro-characterization of active optical devices is outlined. It contains structural as well as functional investigations by means of CT, LM and SEM. The method is demonstrated for a white LED which can be still be operated during characterization.

Video: In Depth Analyses of LEDs by a Combination of X-ray Computed Tomography CT and Light Microscopy LM Correlated with Scanning Electron Microscopy SEM

Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over large or small sample areas. It has been applied in many areas of science, including materials science, geoscience, studying works of cultural heritage, and in chemical biology. In the case of chemical biology, for example, visualizing the metal distributions within cells allows us to study both naturally-occurring metal ions in the cells, as well as exogenously-introduced metals such as drugs and nanoparticles. Due to the fully hydrated nature of nearly all biological samples, cryo-fixation followed by imaging under cryogenic temperature represents the ideal imaging modality currently available. However, under the circumstances that such a combination is not easily accessible or practical, aldehyde based chemical fixation remains useful and sometimes inevitable. This article describes in as much detail as possible in the preparation of adherent mammalian cells by chemical fixation for X-ray fluorescent imaging.

Here, we present a protocol on how to determine the quantity and distribution of metals in a sample using synchrotron X-ray fluorescence. We focus on adherent cells, and describe the chemical fixation method to prepare this sample. We then describe how to mount and image the sample using synchrotron X-rays.

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over ...

Chemistry

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 ...

Failure Analysis of Batteries Using Synchrotron-based Hard X-ray Microtomography

Imaging morphological changes that occur during the lifetime of rechargeable batteries is necessary to understand how these devices fail. Since the advent of lithium-ion batteries, researchers have known that the lithium metal anode has the highest theoretical energy density of any anode material. However, rechargeable batteries containing a lithium metal anode are not widely used in consumer products because the growth of lithium dendrites from the anode upon charging of the battery causes premature cell failure by short circuit. Lithium dendrites can also form in commercial lithium-ion batteries with graphite anodes if they are improperly charged. We demonstrate that lithium dendrite growth can be studied using synchrotron-based hard X-ray microtomography. This non-destructive imaging technique allows researchers to study the growth of lithium dendrites, in addition to other morphological changes inside batteries, and subsequently develop methods to extend battery life.

Synchrotron-based hard X-ray microtomography is used to image the electrochemical growth of dendrites from a lithium metal electrode through a solid polymer electrolyte membrane.

Imaging morphological changes that occur during the lifetime of rechargeable batteries is necessary to understand how these devices fail. Since the advent ...

Using Synchrotron Radiation Microtomography to Investigate Multi-scale Three-dimensional Microelectronic Packages

Synchrotron radiation micro-tomography (SR&#181;T) is a non-destructive three-dimensional (3D) imaging technique that offers high flux for fast data acquisition times with high spatial resolution. In the electronics industry there is serious interest in performing failure analysis on 3D microelectronic packages, many which contain multiple levels of high-density interconnections. Often in tomography there is a trade-off between image resolution and the volume of a sample that can be imaged. This inverse relationship limits the usefulness of conventional computed tomography (CT) systems since a microelectronic package is often large in cross sectional area 100-3,600 mm2, but has important features on the micron scale. The micro-tomography beamline at the Advanced Light Source (ALS), in Berkeley, CA USA, has a setup which is adaptable and can be tailored to a sample&#39;s properties, i.e., density, thickness, etc., with a maximum allowable cross-section of 36 x 36 mm. This setup also has the option of being either monochromatic in the energy range ~7-43 keV or operating with maximum flux in white light mode using a polychromatic beam. Presented here are details of the experimental steps taken to image an entire 16 x 16 mm system within a package, in order to obtain 3D images of the system with a spatial resolution of 8.7 &#181;m all within a scan time of less than 3 min. Also shown are results from packages scanned in different orientations and a sectioned package for higher resolution imaging. In contrast a conventional CT system would take hours to record data with potentially poorer resolution. Indeed, the ratio of field-of-view to throughput time is much higher when using the synchrotron radiation tomography setup. The description below of the experimental setup can be implemented and adapted for use with many other multi-materials.

For this study synchrotron radiation micro-tomography, a non-destructive three-dimensional imaging technique, is employed to investigate an entire microelectronic package with a cross-sectional area of 16 x 16 mm. Due to the synchrotron&#39;s high flux and brightness the sample was imaged in just 3 min&#160;with an 8.7 &#181;m spatial resolution.

Synchrotron radiation micro-tomography (SR&#181;T) is a non-destructive three-dimensional (3D) imaging technique that offers high flux for fast data ...

Comprehensive Characterization of Extended Defects in Semiconductor Materials by a Scanning Electron Microscope

Extended defects such as dislocations and grain boundaries have a strong influence on the performance of microelectronic devices and on other applications of semiconductor materials. However, it is still under debate how the defect structure determines the band structure, and therefore, the recombination behavior of electron-hole pairs responsible for the optical and electrical properties of the extended defects. The present paper is a survey of procedures for the spatially resolved investigation of structural and of physical properties of extended defects in semiconductor materials with a scanning electron microscope (SEM). Representative examples are given for crystalline silicon. The luminescence behavior of extended defects can be investigated by cathodoluminescence (CL) measurements. They are particularly valuable because spectrally and spatially resolved information can be obtained simultaneously. For silicon, with an indirect electronic band structure, CL measurements should be carried out at low temperatures down to 5 K due to the low fraction of radiative recombination processes in comparison to non-radiative transitions at room temperature. For the study of the electrical properties of extended defects, the electron beam induced current (EBIC) technique can be applied. The EBIC image reflects the local distribution of defects due to the increased charge-carrier recombination in their vicinity. The procedure for EBIC investigations is described for measurements at room temperature and at low temperatures. Internal strain fields arising from extended defects can be determined quantitatively by cross-correlation electron backscatter diffraction (ccEBSD). This method is challenging because of the necessary preparation of the sample surface and because of the quality of the diffraction patterns which are recorded during the mapping of the sample. The spatial resolution of the three experimental techniques is compared.

The optical, electrical, and structural properties of dislocations and of grain boundaries in semiconductor materials can be determined by experiments performed in a scanning electron microscope. Electron microscopy has been used to investigate cathodoluminescence, electron beam induced current, and diffraction of backscattered electrons.

Extended defects such as dislocations and grain boundaries have a strong influence on the performance of microelectronic devices and on other applications ...

Enhanced Electron Injection and Exciton Confinement for Pure Blue Quantum-Dot Light-Emitting Diodes by Introducing Partially Oxidized Aluminum Cathode

Stable and efficient red (R), green (G), and blue (B) light sources based on solution-processed quantum dots (QDs) play important roles in next-generation displays and solid-state lighting technologies. The brightness and efficiency of blue QDs-based light-emitting diodes (LEDs) remain inferior to their red and green counterparts, due to the inherently unfavorable energy levels of different colors of light. To solve these problems, a device structure should be designed to balance the injection holes and electrons into the emissive QD layer. Herein, through a simple autoxidation strategy, pure blue QD-LEDs which are highly bright and efficient are demonstrated, with a structure of ITO/PEDOT:PSS/Poly-TPD/QDs/Al:Al2O3. The autoxidized Al:Al2O3 cathode can effectively balance the injected charges and enhance radiative recombination without introducing an additional electron transport layer (ETL). As a result, high color-saturated blue QD-LEDs are achieved with a maximum luminance over 13,000 cd m-2, and a maximum current efficiency of 1.15 cd A-1. The easily controlled autoxidation procedure paves the way for achieving high-performance blue QD-LEDs.

A protocol is presented for fabricating high-performance, pure blue ZnCdS/ZnS-based quantum dots light-emitting diodes by employing an autoxidized aluminum cathode.

Stable and efficient red (R), green (G), and blue (B) light sources based on solution-processed quantum dots (QDs) play important roles in next-generation ...

Synchrotron X-ray Microdiffraction and Fluorescence Imaging of Mineral and Rock Samples

In this report, we describe a detailed procedure for acquiring and processing&#160;x-ray microfluorescence (&#956;XRF), and Laue and powder microdiffraction two-dimensional (2D) maps at beamline 12.3.2 of the Advanced Light Source (ALS), Lawrence Berkeley National Laboratory. Measurements can be performed on any sample that is less than 10 cm x 10 cm x 5 cm, with a flat exposed surface. The experimental geometry is calibrated using standard materials (elemental standards for XRF, and crystalline samples such as Si, quartz, or Al2O3 for diffraction). Samples are aligned to the focal point of the x-ray microbeam, and raster scans are performed, where each pixel of a map corresponds to one measurement, e.g., one XRF spectrum or one diffraction pattern. The data are then processed using the in-house developed software XMAS, which outputs text files, where each row corresponds to a pixel position. Representative data from moissanite and an olive snail shell are presented to demonstrate data quality, collection, and analysis strategies.

We describe a beamline setup meant to carry out rapid two-dimensional x-ray fluorescence and x-ray microdiffraction mapping of single crystal or powder samples using either Laue (polychromatic radiation) or powder (monochromatic radiation) diffraction. The resulting maps give information about strain, orientation, phase distribution, and plastic deformation.

In this report, we describe a detailed procedure for acquiring and processing&#160;x-ray microfluorescence (&#956;XRF), and Laue and powder ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Bioengineering

Light-Induced In Situ Transmission Electron Microscopy for Observation of the Liquid-Soft Matter Interaction

The current protocol describes the modifications of the transmission electron microscope (TEM) setup for in situ light-induced observations. A glass optical fiber inserted into the electron column above the objective lens polepiece, and a laser, an adjustable light source, was used to fabricate the device. After the illuminator has been calibrated using an external measuring system, it allows one to adjust the intensity of the lighting to the needs of the observed process. This lighting system was utilized to image antimicrobial photodynamic therapy phenomena, which are currently the subject of intense research. The sample was prepared by spotting a suspension of bacteria on a carbon, graphene, or silicon nitride substrate, blotting the excess solution, spotting the photosensitizer solution, blotting the excess liquid again, and then assembling the liquid cell with a second substrate or graphene film. The process of the imaging experiment itself includes choosing the right place for observation with the use of low magnification and a minimum dose of electrons, and then cyclical activation of the light source to capture subsequent images at specified intervals with the minimum amount of electrons necessary. The electron dose of each exposure and the time and intensity of lighting used need to be carefully recorded due to the complexity of the observed phenomena as, at the same time, the process is both light- and electron-driven. After the actual experiment is performed, additional control observations must be made, in which the same doses of electrons are used but without additional light influence and smaller doses of electrons are used for higher doses of light. This makes it possible to distinguish light-induced microstructural effects from those caused by electrons in both the fields of life and materials science.

Light-Induced In Situ Transmission Electron Microscopy for Observation of the Liquid-Soft Matter Interaction

The present protocol describes transmission electron microscope (TEM) modifications with a light illumination system, the fabrication of liquid cells, and in situ TEM observations of light-induced interactions between bacterial cells and a photosensitizer. The sample preparation methods, electron beam damage, and imaging are also discussed.

The current protocol describes the modifications of the transmission electron microscope (TEM) setup for in situ light-induced observations. A glass ...

High Spatial Resolution Chemical Imaging of Implant-Associated Infections with X-ray Excited Luminescence Chemical Imaging Through Tissue

Microbial infections associated with implantable medical devices are a major concern in fracture fixation failure. Early diagnosis of such infection will allow successful eradication with antibiotics without an extra cost for a second surgery. Herein, we describe XELCI as a technique with high X-ray resolution, implant specificity, and chemical sensitivity to noninvasively image chemical concentrations near the surface of implanted medical devices. The devices are coated with chemically reporting surfaces. This chemically responsive surface consists of two layers coated on an implantable medical device; a pH-sensitive layer (bromothymol blue or bromocresol green incorporated hydrogel) which is coated over a red-light emitting scintillator (Gd2O2S: Eu) layer for monitoring. A focused X-ray beam irradiates a spot on the implant, and the red light generated by the scintillator (with 620 nm and 700 nm peaks) is transmitted through the sensing layer which alters the spectral ratio depending on the pH. An image is generated by scanning the X-ray beam across the implant and measuring the spectral ratio of light passing through the tissue point-by-point. We used this imaging technique for monitoring implant-associated infections previously on the bone surface of the femur with a modified implantable plate sensor. Now we are studying pH changes that occur from tibial intramedullary rod infections. Two different types of intramedullary rod designs are used in pre-pilot rabbit studies, and we learned that the XELCI technique could be used to monitor any chemical changes that occur not only on the bone surface but also inside the bone. Thus, this enables noninvasive, high spatial resolution, low background local pH imaging to study implant-associated infection biochemistry.

Here, we present a protocol for high-resolution optical detection of chemical information around implanted medical devices with X-ray excited luminescence chemical imaging (XELCI). This novel imaging technique is developed in our lab which enables studying implant-associated infection biochemistry.

Microbial infections associated with implantable medical devices are a major concern in fracture fixation failure. Early diagnosis of such infection will ...

In Depth Analyses of LEDs by a Combination of X-ray Computed Tomography (CT) and Light Microscopy (LM) Correlated with Scanning Electron Microscopy (SEM)

Summary

Explore More Videos

Chapters in this video

Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

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

Failure Analysis of Batteries Using Synchrotron-based Hard X-ray Microtomography

Using Synchrotron Radiation Microtomography to Investigate Multi-scale Three-dimensional Microelectronic Packages

Comprehensive Characterization of Extended Defects in Semiconductor Materials by a Scanning Electron Microscope

Enhanced Electron Injection and Exciton Confinement for Pure Blue Quantum-Dot Light-Emitting Diodes by Introducing Partially Oxidized Aluminum Cathode

Synchrotron X-ray Microdiffraction and Fluorescence Imaging of Mineral and Rock Samples

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

Light-Induced In Situ Transmission Electron Microscopy for Observation of the Liquid-Soft Matter Interaction

High Spatial Resolution Chemical Imaging of Implant-Associated Infections with X-ray Excited Luminescence Chemical Imaging Through Tissue