The LLNL portion of this work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344. The Intel Corporation authors would like to thank Pilin Liu, Liang Hu, William Hammond, and Carlos Orduno from Intel Corporation for some of the data collection and helpful discussions. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.

Note: Protocol details described below were written specifically for work at beamline 8.3.2 at the ALS, Berkeley, CA. Adaptations may be required for work at other synchrotron facilities, which can be found around the world. Appropriate safety and radiation training is required for running experiments at these facilities and the guidelines for training can be found on each individual synchrotron facility&#39;s website. Any changes or updates to the tomography protocol (ALS, LBNL, Berkeley, CA) can be found on the beamline manual15. Details on the tomography process can be found in Reference16. The beamline scientists are available to answer any questions and will facilitate the experimental setup.
1. Steps for Performing Tomography Scans at Beamline 8.3.2 (ALS, LBNL)
<ol>
	<li>Prepare the sample for the scan by mounting it on a sample holder designed to fit in the beamline&#39;s rotational stage. For samples that do not have a custom mount, adhere the sample to a post or drill chuck with clay or wax. 
	Note: The sample scanned in this study was a microelectronic package that is 16 x 16 mm and only ~3 mm in height. In order to fit the entire package in the field of view the sample was mounted horizontally using clay provided at the beamline.
	<ol>
		<li>Align the sample to ensure that when it rotates through 180 degrees it stays within the field of view. Before loading the sample on the rotational stage inside the hutch there is an offline mock rotation stage that is used to align the sample. Visual inspection of the center of rotation is usually sufficient for the alignment.</li>
		<li>Mount the sample attached to the sample holder inside the hutch. Once the sample has been mounted in the hutch, two orthogonal centering motors allow positioning of the sample with respect to the center of rotation. 
		Note: Sometimes sample preparation is needed ahead of experiment time in order to make sure the sample size is correct for the desired resolution. For example, some of the 16 x 16 mm microelectronic packages were sectioned into smaller pieces for further high resolution scanning. The sample size can be determined using Tables 1 and 2.</li>
	</ol>
	</li>
	<li>Select the magnification for the scan based on the sample size and feature size of interest. Beamline 8.3.2 has several lenses to choose from which produce images with a range of pixel sizes from 0.35 - 9 &#181;m. Depending on the magnification, the sample must be of appropriate cross-sectional area, as the field of view decreases with increasing magnification.
	<ol>
		<li>Since the sample scanned here is 22.6 mm in the longest direction, select the 1X lens with the PCO.4,000, as shown in Tables 1 and 2, this combination gives the largest sample field of view. The resulting pixel size is 8.7 &#181;m.</li>
	</ol>
	</li>
	<li>Set the x-ray energy or switch to a polychromatic beam using the Beamline Control Computer. The x-ray energy range at beamline 8.3.2 is continuous from 4-80 keV, but the multilayer monochromator mounted limits the energy range to ~7-43 keV, while the peak flux occurs at ~12 keV. To get the best quality image, base the energy selection on targeting a ~30% transmission, which can be measured on the Data Acquisition Computer. In general, %Transmission increases with increasing energy.
	<ol>
		<li>For the microelectronic package, &#34;select white&#34; light due to the thickness and material of the package. 
		Note: The beamline 8.3.2 manual has detailed steps for changing between &#34;white&#34; light and monochromatic mode.</li>
		<li>When using &#34;white&#34; light mode, add 2-4 metal aluminum and copper filters in line with the x-ray beam in order to filter out the lower energy x-rays. For this sample, use 2 copper sheets with a total thickness of ~1.2 mm.</li>
		<li>Calculate transmission through the sample ahead of time by using: 
		http://henke.lbl.gov/optical_constants/filter2.html or http://www.nist.gov/pml/data/xraycoef/ or http://11bm.xray.aps.anl.gov/absorb/absorb.php. For example, inputting the chemical formula and estimated thickness for the sample will output a graph showing the percent transmission as a function of energy range.</li>
	</ol>
	</li>
	<li>Verify that the stage&#39;s center of rotation is aligned with the camera&#39;s center. To check that the sample is aligned rotate it through 180 degrees using software on the Beamline Control Computer and visually observe the change in sample location by viewing the radiographs on the computer. Control changes to alignment on the same computer. Image quality deteriorates when the sample&#39;s alignment is off enough so that regions of the sample leave the field of view during the sample rotation.</li>
	<li>Manually set sample to detector distance for scan. The camera is on a translational stage that can move horizontally which is used to change the sample to detector distance. When the distance increases the phase contrast contribution also increases. Phase effects are helpful to more easily image subtle cracks and edges, but also cause other &#34;halo effect&#34; artifacts that are often undesirable.</li>
	<li>Verify the beamline alignment. Check the focus of the image and adjust the focus motor if necessary. Confirm that the pixel size calibration is correct by moving the sample a defined amount and measuring the number of pixels the sample moved to calculate &#181;m/pixel. The voxel size will change depending on the experimental setup.
	<ol>
		<li>Check that as the image moves horizontally, image features track horizontally along a constant pixel, and if not, adjust the camera tilt motor so that they do. This aligns the axis of rotation so that it is parallel to pixel columns, which is the alignment assumed later by the reconstruction algorithms.</li>
	</ol>
	</li>
	<li>Select an exposure time for each radiograph. The range for exposure time is 1-1,500 msec and the selection depends on the scan energy and resolution (which determines the observed flux per resolution element). The selected time should provide a tradeoff between the quickest scan time and a scan with more counts and thus the best signal-to-noise ratio.
	<ol>
		<li>For the microelectronic package, use a sample scan time of 100 msec per exposure. 
		Note: Make sure there are no saturated pixels or at least less than the recommended target of 100. The control system is set to display camera counts on a converted scale so that each camera&#39;s maximum counts is 65,535.</li>
	</ol>
	</li>
	<li>Set up the Scan Parameters using the Data Acquisition Computer.
	<ol>
		<li>Input the desired angular range, and the number of images to collect over that range. The more angles selected the longer the scan times and larger the dataset size. Common numbers of angles are 513, 1,025, and 2,049 over a 0-180 degree range. For this study, use 1,025 angles over 180 degrees during data acquisition.</li>
		<li>Select the scanning mode. The two options for scanning mode are 1) normal and 2) continuous tomography. The continuous mode is preferred since it results in the shortest scan time, ~3 min. In this mode, the rotation stage continuously moves as images are collected. In normal mode, the rotation stage stops at each angle and then an image is collected.</li>
		<li>Specify number of bright and dark field images. The bright and dark field images are necessary for performing reconstruction. For the dark field images the shutters close and for the bright field or background images the samples moves out of the field of view. Verify that the sample is translated far enough so that it is not present in the bright field image in order to avoid large defects in the reconstructed images. Here, acquire 15 dark field images and 15 bright field images.</li>
		<li>Determine if tiling is necessary. If the sample is taller than the field of view there is a tiling option, which will scan the sample then translate it vertically until the entire sample is captured.</li>
	</ol>
	</li>
	<li>Execute run scan on the Data Acquisition Computer. The scan will run automatically based on the inputted settings.</li>
</ol>
2. Steps for Performing Tomographic Data Processing
<ol>
	<li>Transfer data to an analysis computer available at the beamline to perform the reconstruction and filtering of the data set using beamline protocol. Reconstruction can run independent of the data acquisition. 
	Note: Data is automatically transferred to NERSC, a high performance computer, where it is processed and reconstructed. Users can sign up for an account at NERSC to access their data through the SPOT Suite web portal at spot.nersc.gov. This portal is still in development mode, so many users prefer to have more control over the reconstruction parameters, in which case they follow the remaining steps.</li>
	<li>Reconstruct the raw images following these steps: 1) normalize images, 2) create stack of sinograms, 3) apply ring removal/filters, and 4) perform parallel beam reconstruction. The reconstruction is based on a filtered back projection algorithm. The reconstruction process results in TIFF images that contain information on the location and intensity of each pixel making up the sample volume. A schematic of the entire process is shown in Figure 2.
	<ol>
		<li>To access the plugin start FIJI (which is an acronym for Fiji Is Just ImageJ) and select the menu Plugins &#8594; ALSmicroCT &#8594; NormalizeStack832newnaming as shown below. A user at the ALS facility can perform the entire reconstruction process using a custom plugin for ImageJ/Fiji, which integrates several software packages designed to streamline the reconstruction process. 
		Note: Fiji and the plugin are available for use on multiple Beamline 8.3.2 analysis computers.</li>
		<li>Once the FIJI dialog box is open, as shown below, select the raw file intended for reconstruction. The stack of raw, bright, and dark images should now be loaded.</li>
		<li>Find the center of rotation by clicking &#39;Detect center of rotation&#39;, then to visualize the reconstructed image select &#39;Preview reconstruction&#39;. The value for center of rotation can also be inputted manually and previewed.</li>
		<li>Using this interface there is the option to change the ring removal parameters, the type of image (8, 16, or 32 bit), pixel range, rotation angle of images, and define cropped region. Each new parameter set can be visualized using the &#39;Preview reconstruction&#39; button.</li>
		<li>Once the parameters are selected, reconstruct the entire stack of images by selecting &#39;run&#39;. All the subsequent data files can be found in the specified &#39;Output directory&#39;, the default directory will be in an output file within the raw data folder.</li>
	</ol>
	</li>
	<li>Access raw data from tomography scans from any computer by going to the website http://spot.nersc.gov/, which is the NERSC (LBNL supercomputer) server through the SPOT portal. 
	Note: Each individual researcher must have their own NERSC account to access their specific datasets. A user can setup an account at https://nim.nersc.gov/nersc_account_request.php. At the beamline, each research group is assigned a beamline account. This account is used to access beamline computers, and can also be used to access data directly from the beamline server using Globus Online.</li>
	<li>Visualize the data in both 3D and 2D by loading the stack of 2D reconstructed images into any 3D analysis software. The samples and images presented here use Avizo software to perform the analysis and visualization, which is available to beamline users at any of the beamline 8.3.2 analysis computers.</li>
	<li>After a data set is uploaded into the visualization software perform further data analysis to get quantified information on specific features within the sample. Often datasets are downsampled in order to reduce the output data size. However this can increase the voxel size reducing fidelity, but smooth the image view for easier segmentation.
	<ol>
		<li>Select segment features of interest by thresholding the histogram of the stack of 2D reconstructed slices and assigning a new pixel value to pixels that fall within a specified range.</li>
		<li>Visualize segmented volumes and surfaces. Once features are segmented they are viewed in 3D using Avizo or any preferred visualization software. This allows for 3D surface renderings of specific features, like solder balls at a certain region of interest.</li>
		<li>Quantify features in sample, i.e., crack size, vias, porosity, defects, etc. Once a feature of interest is identified, such as a via or crack, the feature can be segmented and volumetric information on crack width, length, via volume, porosity distribution can be quantified by evaluating the tomographic data set.</li>
		<li>Create a movie of the sample showing the sample in different orientations. Movie 1 shows examples of the different cross-sectional views and volume rendering views for the microelectronic package imaged in the horizontal orientation.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Tian, T., 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+X-ray+microtomography+study+of+3-D+void+growth+induced+by+electromigration+in+eutectic+SnPb+flip-chip+solder+joints.">Quantitative X-ray microtomography study of 3-D void growth induced by electromigration in eutectic SnPb flip-chip solder joints.</a> Scr. Mater. 65, 646-649 (2011).</li><li>Tian, T., 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=Rapid+diagnosis+of+electromigration+induced+failure+time+of+Pb-free+flip+chip+solder+joints+by+high+resolution+synchrotron+radiation+laminography.">Rapid diagnosis of electromigration induced failure time of Pb-free flip chip solder joints by high resolution synchrotron radiation laminography.</a> Appl. Phys. Lett. 99, 082114 (2011).</li><li>Lee, A., Liu, W., Ho, C. E., Subramanian, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchrotron+x-ray+microscopy+studies+on+electromigration+of+a+two-phase+material.">Synchrotron x-ray microscopy studies on electromigration of a two-phase material.</a> J. Appl. Phys. 102, 053507 (2007).</li><li>Sarobol, P., 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=Effects+of+local+grain+misorientation+and+&#946;-Sn+elastic+anisotropy+on+whisker+and+hillock+formation.">Effects of local grain misorientation and &#946;-Sn elastic anisotropy on whisker and hillock formation.</a> J. Mater. Res. 28, 747-756 (2013).</li><li>Sarobol, P., 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=Recrystallization+as+a+nucleation+mechanism+for+whiskers+and+hillocks+on+thermally+cycled+Sn-alloy+solder+films.">Recrystallization as a nucleation mechanism for whiskers and hillocks on thermally cycled Sn-alloy solder films.</a> Mater. Lett. 99, 76-80 (2013).</li><li>Elmer, J., Specht, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Sn+and+In+Solidification+Undercooling+and+Lattice+Expansion+Using+In+Situ+X-Ray+Diffraction.">Measurement of Sn and In Solidification Undercooling and Lattice Expansion Using In Situ X-Ray Diffraction.</a> J. Electron. Mater. 40, 201-212 (2011).</li><li>Elmer, J., Specht, E., Kumar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microstructure+and+In+Situ+Observations+of+Undercooling+for+Nucleation+of+&#946;-Sn+Relevant+to+Lead-Free+Solder+Alloys.">Microstructure and In Situ Observations of Undercooling for Nucleation of &#946;-Sn Relevant to Lead-Free Solder Alloys.</a> J. Electron. Mater. 39, 273-282 (2010).</li><li>Gourlay, C. M., 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=In+situ+investigation+of+unidirectional+solidification+in+Sn-0.7Cu+and+Sn-0.7Cu-0.06Ni.">In situ investigation of unidirectional solidification in Sn-0.7Cu and Sn-0.7Cu-0.06Ni.</a> Acta Mater. 59, 4043-4054 (2011).</li><li>Ma, H. T., 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=In-situ+study+on+growth+behavior+of+Ag3Sn+in+Sn-3.5Ag/Cu+soldering+reaction+by+synchrotron+radiation+real-time+imaging+technology.">In-situ study on growth behavior of Ag3Sn in Sn-3.5Ag/Cu soldering reaction by synchrotron radiation real-time imaging technology.</a> J. Alloys Compd. 537, 286-290 (2012).</li><li>Zhou, B., 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=In+Situ+Synchrotron+Characterization+of+Melting,+Dissolution,+and+Resolidification+in+Lead-Free+Solders.">In Situ Synchrotron Characterization of Melting, Dissolution, and Resolidification in Lead-Free Solders.</a> J. Electron. Mater. 41, 262-272 (2012).</li><li>Elmer, J., Specht, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-Situ+X-Ray+Diffraction+Observations+of+Low-Temperature+Ag-Nanoink+Sintering+and+High-Temperature+Eutectic+Reaction+with+Copper.">In-Situ X-Ray Diffraction Observations of Low-Temperature Ag-Nanoink Sintering and High-Temperature Eutectic Reaction with Copper.</a> Metall. Mater. Trans. A. 43, 1528-1537 (2012).</li><li>Li, Y., Moore, J. S., Pathangey, B., Dias, R. C., Goyal, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lead-Free+Solder+Joint+Void+Evolution+During+Multiple+Subsequent+High-Temperature+Reflows.">Lead-Free Solder Joint Void Evolution During Multiple Subsequent High-Temperature Reflows.</a> IEEE Trans. Device Mater. Rel. 12, 494-500 (2012).</li><li>Elmer, J., 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=Synchrotron+Radiation+Microtomography+for+Large+Area+3D+Imaging+of+Multilevel+Microelectronic+Packages.">Synchrotron Radiation Microtomography for Large Area 3D Imaging of Multilevel Microelectronic Packages.</a> J. Electron. Mater. 43, 4421-4427 (2014).</li><li>Li, 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=High+Resolution+and+Fast+Throughput-time+X-ray+Computed+Tomography+for+Semiconductor+Packaging+Applications.">High Resolution and Fast Throughput-time X-ray Computed Tomography for Semiconductor Packaging Applications.</a> Proceedings of the 64th IEEE Electronic.Components and Technology Conference (ECTC). , 1457-1463 (2014).</li><li>McElrone, A. J., Choat, B., Parkinson, D. Y., MacDowell, A. A., Brodersen, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+High+Resolution+Computed+Tomography+to+Visualize+the+Three+Dimensional+Structure+and+Function+of+Plant+Vasculature.">Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature.</a> J Vis Exp. , e50162 (2013).</li><li>Kinney, J. H., Nichols, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-Ray+Tomographic+Microscopy+(XTM)+Using+Synchrotron+Radiation.">X-Ray Tomographic Microscopy (XTM) Using Synchrotron Radiation.</a> Annu. Rev. Mater. Sci. 22, 121-152 (1992).</li></ol>

The authors have nothing to disclose.

All of the steps described in the protocol section are critical to obtaining high-resolution images of multi-scale and multi-material samples. One of the most critical steps is the sample mounting and the focusing of optics, which are vital to obtaining quality images that can be used for quantification. Specifically, even slight movement of the sample would cause artifacts in the reconstructed image and defocusing would cause deterioration in resolution. To avoid issues with image quality it is important to reconstruct ...

In the microelectronics field, as in many other fields, non-destructive evaluation at the micrometer scale is necessary when characterizing samples. Specifically for the microelectronics industry there is interest in probing 3D microelectronics packages, containing multi-levels and multi-materials, and identifying failures in packages during thermal, electrical, and mechanical stressing of components. Around the world synchrotron radiation facilities have designated tomography and diffraction beamlines that are used for failure analysis of microelectronic packages. Some examples of this are imaging void formation caused by electromigration1-3, evaluating mechanisms for tin whisker growth4,5, in situ observations of undercooling and anisotropic thermal expansion of tin and intermetallic compounds (IMCs)6,7, in situ observation of solidification and IMC formation8-10, anisotropic mechanical behavior and recrystallization of tin and lead free solders10, voids in flip chip bumps, and in situ observations of Ag-nanoink sintering11. All of these studies have further advanced the understanding and development of components in the microelectronic industry. However, many of these studies have focused on small regions within the package. More information could be gleaned from testing and characterizing the full size package using high resolution SR&#181;T in order to further their development.
The electronic packages being produced now contain multiple layers of interconnects. These packages and devices are growing more and more complex which calls for a 3D solution for non-destructive evaluation with regard to failure analysis, quality control, reliability risk assessment, and development. Certain defects require a technique that can detect features less than 5 &#181;m in size, which include voids and cracks forming inside copper substrate vias, identifying non-contact open and nonwet solder pads in multilevel packaging12, locating and quantifying voids in ball grid arrays (BGAs) and C4 solder joints. During the substrate assembly process these types of defects must be identified and monitored extensively to avoid unwanted failures.
Currently CT systems using laboratory-based sources, also known as tabletop, are able to provide as high as ~1 &#181;m spatial resolution, and are being used to isolate failures in multilevel packages with promising results. However, tabletop CT systems have some limitations when compared to SR&#181;T setups13,14. Tabletop systems are limited to only imaging a certain density range of materials since they usually only contain one or two x-ray source spectrums. Also through-put-time (TPT) remains long for conventional tabletop CT systems requiring several hours of data acquisition time per 1-2 mm2 region of interest, which can limit its usefulness; for instance, analyzing failures in Through Silicon Vias (TSV), BGAs or C4 joints often require acquiring multiple Field of Views (FoV) or regions of interest at high resolution within the sample, resulting in total TPT of 8-12 hours, which is a show stopper for conventional tabletop CT systems when multiple samples have to be analyzed. Synchrotron radiation provides much higher flux and brightness than conventional x-ray sources, resulting in much faster data acquisition times for a given region of interest. Although SR&#181;T does allow for more flexibility with respect to types of materials that can be imaged and sample volume, it does have limitations, which are specific to the synchrotron source and setup used, specifically maximum acceptable thickness and sample size. For the SR&#181;T setup at the ALS the maximum cross-sectional area that can be imaged is &#60;36 x 36 mm and the thickness is limited by the energy range and flux available and is material specific.
This study is used to demonstrate how SR&#181;T can be utilized to image an entire multi-level system in package (SIP) with high resolution and low TPT (3-20 min) for use in inspecting 3D semiconductor packages. More details on comparing tabletop CT&#39;s to Synchrotron Source CT&#39;s can be found in references13,14.
Experimental Overview &#38; Beamline 8.3.2 Description: 
There are synchrotron facilities available for tomography experiments around the world; most of these facilities require submission of a proposal where the experimentalist describes the experiment, as well as its scientific impact. The experiments described here were all performed at the ALS at Lawrence Berkeley National Laboratory (LBNL) at beamline 8.3.2. For this beamline there are two energy mode options: 1) monochromatic in the energy range ~7-43 keV or 2) polychromatic &#34;white&#34; light where the entire available energy spectrum is used when scanning high density materials. During a typical scan at beamline 8.3.2 a sample is mounted on a rotational stage where x-rays penetrate the sample, then the attenuated x-rays are converted into visible light through a scintillator, magnified by a lens, and then projected onto a CCD for recording. This is done while the sample rotates from 0 to 180&#176; producing a stack of images that is reconstructed to obtain a 3D view of the sample with micrometer resolution. The resulting tomographic dataset size ranges from ~3-20 Gb depending on the scan parameters. Figure 1 shows a schematic of the hutch where the sample is scanned.
The following protocol presented here describes the experimental setup, data acquisition, and processing steps required for imaging an entire microelectronic package, but the steps can be modified to image a variety of samples. The modifications depend on the sample size, density, geometries, and features of interest. Tables 1 and 2 present the resolution and sample size combinations available at beamline 8.3.2 (ALS, LBNL, Berkeley, CA). For the microelectronic package investigated here the sample was imaged using a polychromatic (&#34;white&#34;) beam, which was selected due to the thickness and high-density of the sample&#39;s components. The sample was mounted in the horizontal orientation on a chuck mount, this orientation allowed for the entire sample to fit within the height of the beam, which is parallel with a height of ~4 mm and width of ~40 mm, therefore only requiring one scan to capture the entire sample.

<table><tbody><tr><td>Beamline 8.3.2</td><td>Advanced Light Source, Berkeley, CA, USA</td><td></td><td>http://microct.lbl.gov/</td></tr></tbody></table>

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.

The images captured using tomography occur due to the differential absorption of x-rays in the solder interconnects, metallic traces, and other materials in the microelectronic package as a function of the different attenuation lengths and thickness of these multi-materials. The SIP package consisted of a silicon die attached to a ceramic substrate with first level interconnect (FLI) flip chip C4 solder balls of approximately 80 &#181;m diameter; mid-level interconnect (MLI) solder balls of approximately 350 &#181;m conn...

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

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 ...

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10.0K Views.  Lawrence Livermore National Laboratory.  Voor deze studie synchrotronstraling micro-tomografie, een niet-destructieve driedimensionale beeldvormende techniek wordt gebruikt om een ​​volledige micro-elektronische verpakking met een doorsnede van 16 x 16 mm onderzocht. Vanwege de hoge flux en de helderheid van het synchrotron van het monster werd in beeld gebracht in slechts 3 minuten met een 8,7 micrometer ruimtelijke resolutie. 

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

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 ...

Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different size scales. In micrometer and millimeter scales, optical microscopy modalities find increasing use owing to the non-ionizing nature of visible light, and the availability of a rich set of illumination sources (such as lasers and light-emitting-diodes) and detection elements (such as large format CCD and CMOS detector-arrays). Among the recently developed optical tomographic microscopy modalities, one can include optical coherence tomography, optical diffraction tomography, optical projection tomography and light-sheet microscopy. 1-6 These platforms provide sectional imaging of cells, microorganisms and model animals such as C. elegans, zebrafish and mouse embryos.
Existing 3D optical imagers generally have relatively bulky and complex architectures, limiting the availability of these equipments to advanced laboratories, and impeding their integration with lab-on-a-chip platforms and microfluidic chips. To provide an alternative tomographic microscope, we recently developed lensfree optical tomography (LOT) as a high-throughput, compact and cost-effective optical tomography modality. 7 LOT discards the use of lenses and bulky optical components, and instead relies on multi-angle illumination and digital computation to achieve depth-resolved imaging of micro-objects over a large imaging volume. LOT can image biological specimen at a spatial resolution of &lt;1 &mu;m x &lt;1 &mu;m x &lt;3 &mu;m in the x, y and z dimensions, respectively, over a large imaging volume of 15-100 mm3, and can be particularly useful for lab-on-a-chip platforms.

Lensfree optical tomography is a three-dimensional microscopy technique that offers a spatial resolution of &lt;1 &mu;m &times; &lt;1 &mu;m &times; &lt;3 &mu;m in x, y and z dimensions, respectively, over a large imaging-volume of 15-100 mm3, which can be particularly useful for integration with lab-on-a-chip platforms.

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different ...

Bioengineering

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

Biology

Dynamic Pore-scale Reservoir-condition Imaging of Reaction in Carbonates Using Synchrotron Fast Tomography

Underground storage permanence is a major concern for carbon capture and storage. Pumping CO2 into carbonate reservoirs has the potential to dissolve geologic seals and allow CO2 to escape. However, the dissolution processes at reservoir conditions are poorly understood. Thus, time-resolved experiments are needed to observe and predict the nature and rate of dissolution at the pore scale. Synchrotron fast tomography is a method of taking high-resolution time-resolved images of complex pore structures much more quickly than traditional &#181;-CT. The Diamond Lightsource Pink Beam was used to dynamically image dissolution of limestone in the presence of CO2-saturated brine at reservoir conditions. 100 scans were taken at a 6.1 &#181;m resolution over a period of 2 hours. The images were segmented and the porosity and permeability were measured using image analysis and network extraction. Porosity increased uniformly along the length of the sample; however, the rate of increase of both porosity and permeability slowed at later times.

Synchrotron fast tomography was used to dynamically image dissolution of limestone in the presence of CO2-saturated brine at reservoir conditions. 100 scans were taken at a 6.1 &#181;m resolution over a period of 2 h.

Underground storage permanence is a major concern for carbon capture and storage. Pumping CO2 into carbonate reservoirs has the potential to dissolve ...

Visualization of Failure and the Associated Grain-Scale Mechanical Behavior of Granular Soils under Shear using Synchrotron X-Ray Micro-Tomography

The rapid development of X-ray imaging techniques with image processing and analysis skills has enabled the acquisition of CT images of granular soils with high-spatial resolutions. Based on such CT images, grain-scale mechanical behavior such as particle kinematics (i.e., particle translations and particle rotations), strain localization and inter-particle contact evolution of granular soils can be quantitatively investigated. However, this is inaccessible using conventional experimental methods. This study demonstrates the exploration of the grain-scale mechanical behavior of a granular soil sample under triaxial compression using synchrotron X-ray micro-tomography (&#956;CT). With this method, a specially fabricated miniature loading apparatus is used to apply confining and axial stresses to the sample during the triaxial test. The apparatus is fitted into a synchrotron X-ray tomography setup so that high-spatial resolution CT images of the sample can be collected at different loading stages of the test without any disturbance to the sample. With the capability of extracting information at the macro scale (e.g., sample boundary stresses and strains from the triaxial apparatus setup) and the grain scale (e.g., grain movements and contact interactions from the CT images), this procedure provides an effective methodology to investigate the multi-scale mechanics of granular soils.

The protocol describes procedures to acquire high-spatial resolution computed tomography (CT) images of a granular soil during triaxial compression, and to apply image processing techniques to these CT images to explore the grain-scale mechanical behavior of the soil under loading.

The rapid development of X-ray imaging techniques with image processing and analysis skills has enabled the acquisition of CT images of granular soils ...

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.

This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...

Array Tomography Workflow for the Targeted Acquisition of Volume Information using Scanning Electron Microscopy

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission Electron Microscopy (TEM) provided insight into cell ultrastructure, but in the recent decade, the development of modern Scanning Electron Microscopes (SEM) has changed the way of looking inside the cells. Even though the resolution of TEM is superior when protein-level structural details are needed, SEM-resolution is sufficient for the majority of the organelle-level cell biology-related questions. The advancement in technology enabled automatic volume acquisition solutions such as Serial block-face imaging (SBF-SEM) and Focused ion beam SEM (FIB-SEM). Nevertheless, to this day, these methods remain inefficient when the identification and navigation to areas of interest are crucial. Without the means for precise localization of target areas before imaging, operators need to acquire much more data than they need (in SBF-SEM), or, even worse, prepare many grids and image them all (in TEM). We propose the strategy of "lateral screening" using Array Tomography in SEM, which facilitates the localization of areas of interest, followed by automated imaging of the relevant fraction of the total sample volume. Array tomography samples are conserved during imaging, and they can be arranged into section libraries ready for repeated imaging. Several examples are shown in which lateral screening enables us to analyze structural details that are incredibly challenging to access with any other method.

We describe the preparation of ribbons of serial sections and their collection on large transfer support for use as Array Tomography samples, along with automated imaging procedures in a scanning electron microscope. The protocol allows screening, retrieval, and targeted imaging of local, rare events, and the acquisition of large data volumes.

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission ...

Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography

Principles of DNA folding in the cell nucleus and its dynamic transformations that occur during the fulfillment of basic genetic functions (transcription, replication, segregation, etc.) remain poorly understood, partially due to the lack of experimental approaches to high-resolution visualization of specific chromatin loci in structurally preserved nuclei. Here we present a protocol for the visualization of replicative domains in monolayer cell culture in situ,&#160;by combining EdU labeling of newly synthesized DNA with subsequent label detection with Ag-amplification of Nanogold particles and ChromEM staining of chromatin. This protocol allows for the high-contrast, high-efficiency pre-embedding labeling, compatible with traditional glutaraldehyde fixation that provides the best structural preservation of chromatin for room-temperature sample processing. Another advantage of pre-embedding labeling is the possibility to pre-select cells of interest for sectioning. This is especially important for the analysis of heterogeneous cell populations, as well as compatibility with electron tomography approaches to high-resolution 3D analysis of chromatin organization at sites of replication, and the analysis of post-replicative chromatin rearrangement and sister chromatid segregation in the interphase.

This protocol presents a technique for high-resolution mapping of replication sites in structurally preserved chromatin in situ that employs a combination of pre-embedding EdU-streptavidin-Nanogold labeling and ChromEMT.

Principles of DNA folding in the cell nucleus and its dynamic transformations that occur during the fulfillment of basic genetic functions (transcription, ...

Cryo-Electron Tomography Remote Data Collection and Subtomogram Averaging

Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved automated acquisition strategies, preparative techniques that expand the possibilities of what the electron microscope can image at high-resolution using cryo-ET and new subtomogram averaging software. Additionally, data acquisition has become increasingly streamlined, making it more accessible to many users. The SARS-CoV-2 pandemic has further accelerated remote cryo-electron microscopy (cryo-EM) data collection, especially for single-particle cryo-EM, in many facilities globally, providing uninterrupted user access to state-of-the-art instruments during the pandemic. With the recent advances in Tomo5 (software for 3D electron tomography), remote cryo-ET data collection has become robust and easy to handle from anywhere in the world. This article aims to provide a detailed walk-through, starting from the data collection setup in the tomography software for the process of a (remote) cryo-ET data collection session with detailed troubleshooting. The (remote) data collection protocol is further complemented with the workflow for structure determination at near-atomic resolution by subtomogram averaging with emClarity, using apoferritin as an example.

The present protocol describes high-resolution cryo-electron tomography remote data acquisition using Tomo5 and subsequent data processing and subtomogram averaging using emClarity. Apoferritin is used as an example to illustrate detailed step-by-step processes to achieve a cryo-ET structure at 2.86 &#197; resolution.

Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved ...

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

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