Research described in this paper was performed at the BMIT facility at the Canadian Light Source, which is supported by the Canada Foundation for Innovation, Natural Sciences and Engineering Research Council of Canada, the University of Saskatchewan, the Government of Saskatchewan, Western Economic Diversification Canada, the National Research Council Canada, and the Canadian Institutes of Health Research. The authors would like to thank the beamline scientists at the Canadian Light Source, particularly Adam Webb, Denise Miller, Sergey Gasilov, and Ning Zu for the assistance in set-up and troubleshooting of the SkyScan SR&#181;CT and white beam microscope systems. We also wish to thank Beth Dalzell from The University of Toledo College of Medicine and Life Sciences and Dr. Jeffrey Wenstrup of the Northeast Ohio Medical University for access to cadaveric samples for this study. JM Andronowski is supported through start-up research funds provided by The University of Akron and a National&#160;Institute of Justice Research and&#160;Development in Forensic Science&#160;for Criminal Justice Purposes grant (2018-DU-BX-0188).&#160;RA Davis is supported by a graduate assistantship provided by The University of Akron. Equipment and supplies used for coring and sawing were purchased by start-up funds provided by The University of Akron and NSF grant EAR-1624242 to CW Holyoke.

All specimens were sourced from embalmed cadaveric donors at the University of Toledo, College of Medicine and Life Sciences and Northeast Ohio Medical University (NEOMED), with the informed consent of the donor themselves or the donor&#39;s next-of-kin. The University of Akron Institutional Review Board for the Protection of Human Subjects (IRB) deemed these specimens exempt from full IRB review as they were not procured from living individuals. Demographic information including age, sex, and cause of death were available for all donors. The selected individuals did not have documented bone-affecting conditions nor exposure to treatment regimens that may have affected bone remodeling at the time of death. Cortical bone samples were obtained from femora of cadaveric modern males and females with ages ranging from 19 to 101 years of age (mean = 73.9 years). The femoral midshaft has been studied extensively including examinations of variation in cortical porosity20,21,22,23,24 and material density of bone tissue25,26,27, and has thus become a commonly used site for microstructural analyses.
1. Tissue Procurement and Maceration
<ol>
	<li>Use an oscillating saw equipped with a plunge cutting carbide blade (for composite materials) to procure ~7.5 cm bone blocks from the mid-diaphyses of the left femora.</li>
	<li>Soak femoral blocks in an oven-safe glass dish filled with a powdered protease enzyme and tap water solution for 1 h in an incubator set at 45 &#176;C.</li>
	<li>After incubation, carefully remove any remaining soft tissues and periosteum using blunt dissection or dental tools. 
	NOTE: Avoid the use of sharp tools (e.g., scalpel) to remove soft tissues. Such instruments may cause damage to the bone that is detectable in &#181;CT scans, affecting specimen preservation and scan data quality.</li>
	<li>Remove debris or occlusions in the medullary cavity by placing bone blocks in an ultrasonic cleaner for 5-10 minutes with 20:1 parts tap water to cleaning solution (see Table of Materials) or using a handheld water flosser (e.g., Waterpik).</li>
	<li>Immerse the bone block in a specimen cup and fill with 70% ethanol. Allow the bone to soak for at least 24 hours to remove lipids. 
	NOTE: Xylenes may also be used to remove lipids. Extended soaking in xylenes, however, may make the bone brittle or chalky as it is an emulsifier.</li>
	<li>After 24 hours, remove bone blocks from ethanol and allow to air dry at ambient temperature for 24-48 hours. 
	NOTE: The protocol may be paused here.</li>
</ol>
2. Tissue sectioning
<ol>
	<li>Place a 75 x 25 mm glass microscope slide on a hot plate set to 140 &#176;C. Melt a generous amount of thermal epoxy resin (see Table of Materials) on the center of the slide.
	<ol>
		<li>If preparing additional thin sections for microscopy (&#60;50 &#181;m), the bone block may need to be embedded in a two-part epoxy to preserve trabeculae. Further, when implementing this protocol for fragile specimens (e.g., diagenetic bone or highly trabecularized specimens) embedding specimens in an epoxy is necessary. 
		NOTE: Bone samples used in this protocol were retrieved from embalmed cadaveric specimens. If fresh specimens are collected at autopsy or from a surgical case to examine soft tissue structures (e.g., vasculature) via SR&#181;CT, impregnation with epoxy may induce damage to such tissues. In these cases, an alternative adhesive or mounting medium is recommended (e.g., double-sided tape, modeling clay).</li>
	</ol>
	</li>
	<li>Press the inferior aspect of the bone block into the thermal epoxy resin on the microscope slide, with the length of the bone perpendicular to the slide. Shift the sample back-and-forth in order to coat the underside of the bone and ensure secure adhesion to the slide.</li>
	<li>Let the mounted specimen rest on the hot plate for ~5 min to allow the thermal epoxy to wick into pores and/or cracks. 
	NOTE: The epoxy on the slide should be free of bubbles for best adhesion. To remove bubbles, shift the sample back and forth on the slide. Bubbles often form due to water and/or ethanol trapped within the bone escaping and evaporating.</li>
	<li>Remove the slide with the mounted specimen from the hot plate using blunt forceps and allow to cool at room temperature for ~10 minutes. Remove any epoxy from the edge of the slide using a razor blade to ensure the chuck adequately grips the slide.</li>
	<li>Attach the slide with the adhered sample to a glass slide chuck and mount the chuck to the swivel arm of a slow-speed sectioning saw (see Table of Materials, Figure 2). 
	NOTE: While a Buehler IsoMet saw was employed in this protocol, other precision sectioning saws are available that could be used in place of the IsoMet (e.g., Leco, Exakt, Smartcut, CT3, Buehler Petrothin, Well Diamond Wire).</li>
	<li>Adjust the swivel arm using the positioning dial to ensure the blade contacts and transects the sample. Position the specimen such that a cross-section of the bone will be cut perpendicular to its length.</li>
	<li>Add weights to the far side of the cutting arm to counter the weight of the arm. 
	NOTE: If insufficient counterweight is used, the sample may bear down on the blade and cause the blade to fracture.</li>
	<li>Add cutting fluid (20:1 parts water to cutting fluid) to the fluid receptacle of the saw.</li>
	<li>Tightly secure the diamond wafer blade and ensure the fluid level submerges the cutting portion of the blade. Set the speed to 200 RPM and slowly lower the sample onto the blade (Figure 3).</li>
	<li>Ensure the blade and chuck are not wobbling and/or bouncing. If excessive movement is noted, immediately stop the saw and tighten the blade and/or chuck arm assembly before resuming cutting. Add additional counterweights if the chuck is aggressively moving up and down. Excessive movement including visible side-to-side motion may cause the blade to fracture.</li>
	<li>The first thick section is a &#39;waste cut&#39;&#160;to provide a well-defined surface parallel to each additional cut. After the initial waste cut, raise the swivel arm and move the chuck towards the blade 5 mm using the positioning dial. Further thick sections (~1 mm) for microscopy can further be collected with this method. 
	NOTE: In order to save valuable tissue, the waste cut can be omitted. When sectioning a sample with an uneven edge, however, it is critical that the peak of the specimen be lined up tangentially to the edge of the coring drill bit.
	<ol>
		<li>Be sure to account for the kerf of the blade when sectioning. For example, to get a 5 mm section from a blade that has a kerf of 0.5 mm, move the sample and chuck 5.5 mm towards the blade.</li>
	</ol>
	</li>
	<li>After sectioning is complete, place the glass slide with the mounted specimen on a hot plate to melt the thermal epoxy. This allows for swift removal of bone blocks from the slide. 
	NOTE: The protocol can be paused here.</li>
</ol>
3. Sample coring
<ol>
	<li>Mount 5 mm bone sections to the bottom of a shallow aluminum tin (~8 cm in diameter) using the thermal epoxy bonding technique as described in steps 2.2-2.4.</li>
	<li>Place tin on an XY machine table of the mill-drill press (see Table of Materials) and hand tighten fixturing clamps (Figure 4).</li>
	<li>Insert 2 mm inner-diameter hollow-shaft jeweler&#39;s diamond-tipped coring drill bit (see Table of Materials) to the mill-drill chuck. Adjust the depth limiter to prevent coring through the tin (Figure 5).</li>
	<li>Align the central anterior aspect of the bone sample beneath the drill bit while avoiding close contact with either the periosteum, endosteum, or highly trabecularized areas. 
	NOTE: Automated selection of mid-anterior femoral cortices is not feasible as cortical thickness varies among individuals, especially with increasing age.</li>
	<li>Fill the tin with distilled water to completely cover the sample. This prevents heat build-up, burning of the sample, and/or damage to the drill bit during coring. 
	NOTE: To assess the possibility of heat damage caused by coring, an infrared thermometer was used to achieve temperature readings from the distilled water as the coring bit first penetrated the bones&#39;&#160;surface. The temperature varied by 1 &#176;C, from 22.9 &#8211; 23.9 &#176;C among the ten samples cored for this test. Thus, we argue that heat-induced damage is negligible.</li>
	<li>For the first few instances of contact between the core bit and bone, apply gentle pressure in order to wear a ring on the superior surface of the bone. This prevents deflection of the drill bit at the beginning of the coring process and ensures correct placement of the bit.</li>
	<li>During coring, lift the drill bit in and out of the sample while keeping the bit&#39;s tip beneath the water&#8217;s surface. Continue this technique every few seconds to flush out trapped bone dust and ensure debris is not occluding the drill bit. 
	NOTE: If the core is forming a conical shape, it is likely due to 1) allowing insufficient time to flush bone dust from the coring bit, and 2) coring is occurring too quickly. Increased speed may break off large pieces from the sample and pulverize the superior aspect.</li>
	<li>After coring is complete, the resulting bone core may become lodged in the hollow-stemmed drill bit (Figure 6). Use a pair of fine-tipped forceps or a small Allen wrench to dislodge the core from the bit (Figure 2).</li>
	<li>Store the cored sample in a labelled microcentrifuge tube in a cool and dry location until imaging.</li>
</ol>
4. Image processing routines for evaluating bone microarchitectural parameters from cortical bone cores
<ol>
	<li>Reconstruction of &#181;CT images
	<ol>
		<li>Download and install the latest NRecon version at https://www.bruker.com/products/microtomography.html for reconstruction of the SR&#181;CT projection images.</li>
		<li>Select the NRecon shortcut on the Desktop and the associated GPUReconServer will appear.</li>
		<li>Open desired dataset in pop-up window. If window does not appear, select the folder icon on the upper left-hand corner of the dataviewer window.</li>
		<li>Select the first projection from the SR&#181;CT acquisition. Under Output, remove the selections for Use ROI and Scales ON.</li>
		<li>Choose the reconstruction file destination. Select Browse and create a new folder named Recon. The selected file format should be BMP(8).</li>
		<li>Check Misalignment Compensation. 
		NOTE: This estimation is often close to correct. The rough 3D render can be manually adjusted by moving the arrows up and down to shift the overlapping images so that the right and left edges align as closely as possible.</li>
		<li>Under Settings, choose desired selections to apply Smoothing, Beam Hardening, CS Rotation, Object Larger than FOV, and Ring Artifacts algorithms.</li>
		<li>Adjust the histogram under Output by selecting Auto. 
		NOTE: The resulting image may be dim.</li>
		<li>Select Start to begin processing the reconstruction.</li>
		<li>Use standard nomenclature for canal/osteocyte lacunar indices28. These may include: total VOI tissue volume (TV), canal volume (Ca.V), total number of canals (Ca.N), average canal diameter (Ca.Dm), cortical porosity (Ca.V/TV), given as a percentage, total number of lacunae (N.Lc), and average lacunar volume (Lc.V), among others. To determine lacunar density per mm3 (N.Lc/BV), bone volume (BV) is calculated as total volume minus canal volume (TV-Ca.V). 
		NOTE: The protocol can be paused here.</li>
	</ol>
	</li>
	<li>Collection of Microarchitectural Data from Reconstructed Images
	<ol>
		<li>Download and install the latest version of CTAnalyser at https://www.bruker.com/products/microtomography/micro-ct-software/3dsuite.html for analysis of microarchitectural parameters. 
		NOTE: The free version of CTAnalyser is limited in functionality. Therefore, it is recommended to purchase a full license to perform more detailed analyses.</li>
		<li>Under Image | Properties | Change Pixel size, ensure the pixel size matches that of the applied &#181;CT imaging protocol. 
		NOTE: If editing images in ImageJ or a similar program, note that upon saving, the header embedded in the TIFF file will be modified and the analysis software will alter the pixel size when importing the dataset.</li>
		<li>Select Custom Processing in order to create a task list (see Supplementary Materials) to analyze the bone microarchitecture from the scan dataset. A general protocol for osteocyte lacunar network parameters using plugins proprietary to CTAnalyser follows here: 
		NOTE: The plugin task list works well for datasets where the sample is the only subject visible in the FOV. If empty space surrounds the specimen, the application of an ROI is required. Otherwise, the values gathered in 3D Analysis and Individual Object Analysis will be artificially decreased.
		<ol>
			<li>Reload the images to reset and/or adjust any modifications (e.g., from editing in ImageJ or similar) prior to opening the Custom Processing menu in the analysis software.</li>
			<li>To reduce noise in the images, apply a Gaussian low-pass filter in 3D space with a round kernel and a radius of 2-3. 
			NOTE: These settings were applied to datasets from the reported SR&#181;CT experiments through trial and error testing. The goal was to obtain the best quality reconstructions for the data. Adjust reconstruction settings to suit each unique experimental setup.</li>
			<li>Apply a global grayscale threshold to the images by selecting low and high values to highlight vascular canals. The reconstructed slices seen in Figures 8B and 8D depict an example threshold of 0-155. 
			NOTE: Similar to step 4.2.3.2, the threshold settings applied here were chosen through extensive trial and error. Thresholding should be adjusted for each experimental set-up and &#181;CT imaging system used.</li>
			<li>Despeckle (denoise) to remove white speckles in 3D space that are within the volumetric pixel (voxel) size range of osteocyte lacunae in order to isolate canals only. 
			NOTE: For a SR&#181;CT scan of human cortical bone taken at 0.9 &#181;m pixel size, the lower limit for osteocyte lacunae is 13 voxels.</li>
			<li>Despeckle to remove any black speckles in 2D space to remove artifacts in the canals. These can be quite large in 2D, thus remove features that are &#60;15,000 pixels.</li>
			<li>Dilate the pores in 3D space using the Morphological operation function with a round kernel of 2 or 3 radius, depending on quality of images, in order to isolate any soft tissues trapped in canals.</li>
			<li>Perform an additional Despeckle function using the same settings as step 4.2.3.5. in order to remove isolated soft tissues within canals.</li>
			<li>Erode the dilation from step 4.2.3.6. using a Morphological operation function using a round kernel with either 2 or 3 radius. The radius for this step must match the radius used in Procedure 4.2.3.6.</li>
			<li>Run 3D Analysis and select which parameters to calculate for the volume of vascular canals. Generally, the basic values will provide sufficient information.</li>
			<li>Save the processed images with Save Bitmaps into a custom subfolder in the directory. 
			NOTE: If creating a 3D reconstruction image from the processed images using a program such as Amira/Avizo, Dragonfly, Drishti, etc., saving the images as monochrome (1 bit) is recommended.</li>
			<li>Calculate the number of vascular canals and describe their size, shape, and orientation using the Individual Object Analysis function.</li>
			<li>Repeat steps 4.2.3.1 &#8211; 4.2.3.3. to reset the image for osteocyte lacunar analysis.</li>
			<li>Remove white speckles in 3D space using the Despeckle function, ensuring that such artifacts are smaller than the lower limit of lacunar size. This step removes noise from the scan that may appear to be cortical pores, while preserving true osteocyte lacunae. For human SR&#181;CT scans of 0.9 &#181;m pixel size, this lower limit is 13 voxels.</li>
			<li>Despeckle once more to remove white speckles that are larger than the upper limit of lacunar size. For human SR&#181;CT datasets with the settings listed in step 4.2.3.13, this limit is 2743 voxels.</li>
			<li>Perform 3D Analysis to extract microstructural information pertaining to the osteocyte lacunae specifically.</li>
			<li>Select Save Bitmaps to save the processed images in order to isolate the osteocyte lacunae.</li>
			<li>Perform Individual Object Analysis to calculate the number of osteocytes in 3D within the selected Volume of Interest (VOI). 
			NOTE: Once the task list has been established and tested, CTAnalyser has a batch manager (BatMan) function which can be employed to accelerate data extraction and ensure uniform image processing. A task list with example settings for Procedure 4.2.3. can be found in the Supplementary Materials.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+implications+of+porosity+distribution+in+bone+of+the+appendicular+skeleton.">Mechanical implications of porosity distribution in bone of the appendicular skeleton.</a> Orthopedic Transactions. 8, 342-343 (1984).</li><li>Bousson, V., 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=Distribution+of+Intracortical+Porosity+in+Human+Midfemoral+Cortex+by+Age+and+Gender.">Distribution of Intracortical Porosity in Human Midfemoral Cortex by Age and Gender.</a> Journal of Bone and Mineral Research. 16 (7), 1308-1317 (2001).</li><li>Goldman, H. M., Thomas, C. D. L., Clement, J. G., Bromage, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationships+among+microstructural+properties+of+bone+at+the+human+midshaft+femur.">Relationships among microstructural properties of bone at the human midshaft femur.</a> Journal of Anatomy. 206 (2), 127-139 (2005).</li><li>De Micheli, P. O., Witzel, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microstructural+mechanical+study+of+a+transverse+osteon+under+compressive+loading:+The+role+of+fiber+reinforcement+and+explanation+of+some+geometrical+and+mechanical+microscopic+properties.">Microstructural mechanical study of a transverse osteon under compressive loading: The role of fiber reinforcement and explanation of some geometrical and mechanical microscopic properties.</a> Journal of Biomechanics. 44 (8), 1588-1592 (2011).</li><li>Martin, R. B., Boardman, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+collagen+fiber+orientation,+porosity,+density,+and+mineralization+on+bovine+cortical+bone+bending+properties.">The effects of collagen fiber orientation, porosity, density, and mineralization on bovine cortical bone bending properties.</a> Journal of Biomechanics. 26 (9), 1047-1054 (1993).</li><li>Cooper, D. M. L., Turinsky, A. L., Sensen, C. W., Hallgr&#237;msson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+3D+analysis+of+the+canal+network+in+cortical+bone+by+micro-computed+tomography.">Quantitative 3D analysis of the canal network in cortical bone by micro-computed tomography.</a> The Anatomical Record Part B: The New Anatomist. 274 (1), 169-179 (2003).</li><li>Crowder, C., Heinrich, J., Stout, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rib+histomorphometry+for+adult+age+estimation.">Rib histomorphometry for adult age estimation.</a> Forensic Microscopy for Skeletal Tissues: Methods and Protocols. , 109-127 (2012).</li><li>Pfeiffer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paleohistology:+Health+and+disease.">Paleohistology: Health and disease.</a> Biological Anthropology of the Human Skeleton. , 287-302 (2000).</li><li>Bone Hedges, R. E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=diagenesis:+an+overview+of+processes.">diagenesis: an overview of processes.</a> Archaeometry. 44 (3), 319-328 (2002).</li><li>. The use of formaldehyde imbedded human remains in experimental procedures Available from: <a target="_blank" href="https://capa-acap.net/sites/default/files/basic-page/capa_2017_program_final_no_cover.pdf">https://capa-acap.net/sites/default/files/basic-page/capa_2017_program_final_no_cover.pdf</a> (2017)</li><li>Currey, J. D., Brear, K., Zioupos, P., Reilly, G. C. <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+formaldehyde+fixation+on+some+mechanical+properties+of+bovine+bone.">Effect of formaldehyde fixation on some mechanical properties of bovine bone.</a> Biomaterials. 16 (16), 1267-1271 (1995).</li><li>Asaka, T., Kikugawa, H. <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+formaldehyde+solution+on+fracture+characteristics+of+bovine+femoral+compact+bone.">Effect of formaldehyde solution on fracture characteristics of bovine femoral compact bone.</a> Journal of the Japan Institute of Metals. 69 (8), 711-714 (2005).</li></ol>

The authors have nothing to disclose.

There has been no comprehensive, standardized protocol for procuring uniform and cylindrical cortical bone core samples for high-resolution SR&#181;CT imaging with limited FOV setups. The protocol detailed here fills&#160;that void by providing a comprehensive tutorial regarding how to procure consistently sized cortical bone core samples for SR&#181;CT imaging and the subsequent accurate visualization and extraction of microarchitectural data. We have shown that our protocol provides a more standardized and reliable met...

With recent advancements in imaging technology, it is now feasible to acquire X-ray imaging data with very high resolution. Desktop micro-CT (&#181;CT) systems are the current standard for imaging cancellous bone due to their non-destructive nature1. When imaging microstructural features of cortical bone, however, &#181;CT use has been more limited. Due to resolution constraints, desktop systems cannot attain the resolution required to image microstructural features smaller than cortical pores, such as osteocyte lacunae. For this application, SR&#181;CT is ideal owing to the greater resolution of these systems1. For example, experiments at the Canadian Light Source (CLS) on the BioMedical Imaging and Therapy (BMIT) beamlines2 have produced images with voxels as small as 0.9 &#181;m. Previous studies1,3,4,5 have used this resolution to acquire projections and subsequent three-dimensional (3D) renders from cortical bone specimens from human long bones (Figure 1) to quantify osteocyte lacunar density4,6,7,8,9 and variation in the lacunar shape and size3 across the human lifespan and between the sexes. Further studies have demonstrated the presence of osteon banding in humans10, a phenomenon previously recognized to be associated with only nonhuman mammals in the forensic anthropological literature.
In order to achieve exceptional resolution, the X-ray beam must be finely focused within the field of view (FOV), which often limits the maximum specimen size to a few millimeters in diameter. Currently, there have been no comprehensive, standardized procedures described in the literature outlining bone sample procurement that meet these restrictions. Centering specimens within the FOV is critical to ensure that 1) the sample remains centered as it rotates 180&#176; during imaging, and 2) scan artifacts are limited since there is no image truncation. In other words, no portions of the sample outside of the FOV interfere with the beam entering its focal point inside the FOV. If this occurs, the reconstruction algorithm is deprived of some of the attenuation data needed for a fully correct reconstruction. It is further worth noting that 360&#176; (full rotation) scans minimize the effects of beam hardening but increase artifacts caused by misalignment and sample movement during imaging. Thus, while a 360&#176; scan will typically generate cleaner data, imaging time is doubled and so a compromise between experimental cost and data quality must be addressed.
An important and often overlooked aspect of bone imaging experiments is the accurate and replicable specimen preparation technique performed prior to scanning. Studies that incorporate SR&#181;CT methods into their experiments briefly mention their sampling protocol, but the authors provide little to no detail regarding the particular methodology used to gather their specimens. Many such studies mention cutting rectilinear bone blocks of arbitrary dimensions, but generally provide no further information about the tools or embedding materials used3,4,10,11,12,13,14. Some researchers commonly use handheld rotary tools (e.g., Dremel) to remove rectilinear blocks of bone from a region of interest (ROI)3,4,10,11,12,13,14. This method results in nonuniformly sized samples that may be larger than the FOV, increasing the likelihood of scan artifacts and image truncation. Such specimens often require further refining using a precision diamond-wafer saw (e.g., Buehler Isomet). Procuring samples with consistent dimensions (to the two-hundredths/mm) is critical to ensure that the acquired datasets are of the highest quality and the subsequent results are replicable.
The limited reporting of sample procurement methodology adds an extra layer of difficulty when attempting to employ and/or validate methods performed in a previous study. Currently, researchers must contact authors directly for further details on their sampling procedures. The protocol detailed here provides biomedical researchers with a thoroughly documented, replicable, and cost-efficient sampling technique. The primary objective of this article is to provide a comprehensive tutorial regarding how to procure consistently sized cortical bone core samples using a mill-drill press and diamond coring bit for the accurate visualization and extraction of microarchitectural data. This method is modified from procedures used to routinely collect uniform, small-diameter (1-5 mm) cylinders from blocks of hard materials in high pressure rock mechanics15,16,17,18,19.

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a sectioning, coring, and image processing guide for high-throughput cortical bone sample procurement and analysis for synchrotron micro-ct

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been studied substantially using traditional two-dimensional techniques. Recent advancements in X-ray imaging technology via desktop micro-computed tomography (&#181;CT) and synchrotron radiation micro-computed tomography (SR&#181;CT) have allowed for the acquisition of high-resolution three-dimensional (3D) scans of a larger field of view (FOV) than other 3D imaging techniques (e.g., SEM) providing a more complete picture of microscopic structures within human cortical bone. The specimen should be accurately centered within the FOV, however, to limit the appearance of streak artifacts known to impact data analysis. Previous studies have reported procurement of irregularly shaped rectilinear bone blocks that result in imaging artifacts due to uneven edges or image truncation. We have applied a geological sampling protocol (coring) to procure consistently sized cortical bone core specimens for SR&#181;CT experiments from the anterior aspect of human femora. This coring method is efficient and minimally destructive to tissue. It creates uniform cylindrical samples that decrease imaging artifacts by nature of being isometric during rotation and providing a uniform path length for X-ray beams throughout scanning. Image processing of X-ray tomographic data of cored and irregularly shaped samples confirms the potential of the technique to improve visualization and analysis of cortical bone microarchitecture. A goal of this protocol is to deliver a reliable and repeatable method for the extraction of cortical bone cores that is adaptable for various types of high-resolution bone imaging experiments. An overarching goal of the work is to create a standardized cortical bone procurement for SR&#181;CT that is affordable, consistent, and straightforward. This procedure may further be adapted by researchers in related fields who commonly evaluate hard composite materials such as in biological anthropology, geosciences, or material sciences.

The described method of core sampling proved to be highly effective and efficient. Coring specimens using this protocol allowed for the procurement of &#62;300 consistently sized samples for experiments on the CLS BMIT-BM beamline2, with an FOV of ~2 mm at 1.49 &#181;m voxel size. To validate the consistency of core diameter, three measurements were taken along the length (top, middle, bottom) of a subset of human anterior femoral cores (n=69). The average diameter of the cores was 1.96 &...

Watch this Scientific Journal Video about A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT at JoVE.com

A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT

We employed a geological (coring) sampling protocol to procure cortical bone specimens of uniform size for SR&#181;CT experiments from the anterior aspect of human femora. This method is minimally destructive, efficient, results in cylindrical specimens that minimize imaging artifacts from irregular sample shapes and improves microarchitectural visualization and analysis.

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been ...

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5.0K Views.  The University of Akron. We employed a geological (coring) sampling protocol to procure cortical bone specimens of uniform size for SRµCT experiments from the anterior aspect of human femora. This method is minimally destructive, efficient, results in cylindrical specimens that minimize imaging artifacts from irregular sample shapes and improves microarchitectural visualization and analysis.

Video: A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT

A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production 1. Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils 2, buffalo rumen 3 and the termite hind-gut 4 using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood 5). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio 6. Other methods have been reported 7,8 but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate 9. Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction 10. The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

This protocol describes a high throughput screen for cellulolytic activity from a metagenomic library expressed in Escherichia coli. The screen is solution based and highly automated, and uses one-pot chemistry in 384 well microplates with the final readout as an absorbance measurement.

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel ...

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a plethora of mutant strains and technical tricks have been developed to allow sophisticated questions to be asked and answered in a reasonable amount of time. Fundamental insight into the interplay of components of all known major signaling pathways has been obtained in forward and reverse genetic Drosophila studies. The fly eye has proven to be exceptionally well suited for mutational analysis, since, under laboratory conditions, flies can survive without functional eyes. Furthermore, the surface of the insect eye is composed of some 800 individual unit eyes (facets or ommatidia) that form a regular, smooth surface when looked at under a dissecting microscope. Thus, it is easy to see whether a mutation might affect eye development or growth by externally looking for the loss of the smooth surface ('rough eye' phenotype; Fig. 1) or overall eye size, respectively (for examples of screens based on external eye morphology see e.g.1). Subsequent detailed analyses of eye phenotypes require fixation, plastic embedding and thin-sectioning of adult eyes.
The Drosophila eye develops from the so-called eye imaginal disc, a bag of epithelial cells that proliferate and differentiate during larval and pupal stages (for review see e.g. 2). Each ommatidium consists of 20 cells, including eight photoreceptors (PR or R-cells; Fig. 2), four lens-secreting cone cells, pigment cells ('hexagon' around R-cell cluster) and a bristle. The photoreceptors of each ommatidium, most easily identified by their light sensitive organelles, the rhabdomeres, are organized in a trapezoid made up of the six "outer" (R1-6) and two "inner" photoreceptors (R7/8; R8 [Fig. 2] is underneath R7 and thus only seen in sections from deeper areas of the eye). The trapezoid of each facet is precisely aligned with those of its neighbors and the overall anteroposterior and dorsoventral axes of the eye (Fig. 3A). In particular, the ommatidia of the dorsal and ventral (black and red arrows, respectively) halves of the eye are mirror images of each other and correspond to two chiral forms established during planar cell polarity signaling (for review see e.g. 3).
The method to generate semi-thin eye sections (such as those presented in Fig. 3) described here is slightly modified from the one originally described by Tomlinson and Ready4. It allows the morphological analysis of all cells except for the transparent cone cells. In addition, the pigment of R-cells (blue arrowheads in Fig. 2 and 3) can be used as a cell-autonomous marker for the genotype of a R-cell, thus genetic requirements of genes in a subset of R-cells can readily be determined5,6.

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

A standard approach to prepare adult Drosophila eyes for semi-thin sectioning and light microscopic analysis is presented here. The protocol can be used for gross morphological analysis of eye defects, or with the indicated adjustments can be used to determine genetic requirements of genes in specific cell types of the eye (e.g. clonal analysis of photoreceptors) or for electron microscopic analysis.

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a ...

A Practical Guide to Phylogenetics for Nonexperts

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this topic and so it presents inherent problems. Here we compile a practical introduction to phylogenetics for nonexperts.&#160;We outline in a step-by-step manner, a pipeline for generating reliable phylogenies from gene sequence datasets. We begin with a user-guide for similarity search tools via online interfaces as well as local executables. Next, we explore programs for generating multiple sequence alignments followed by protocols for using software to determine best-fit models of evolution. We then outline protocols for reconstructing phylogenetic relationships via maximum likelihood and Bayesian criteria&#160;and finally describe tools for visualizing phylogenetic trees. While this is not by any means an exhaustive description of phylogenetic approaches, it does provide the reader with practical starting information on key software applications commonly utilized by phylogeneticists. The vision for this article would be that it could serve as a practical training tool for researchers embarking on phylogenetic studies&#160;and also serve as an educational resource that could be incorporated into a classroom or teaching-lab.

Here we describe a step-by-step pipeline for generating reliable phylogenies from nucleotide or amino acid sequence datasets. This guide aims to serve researchers or students new to phylogenetic analysis.&#160;

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

Sample Preparation for Mass Cytometry Analysis

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.

This article describes the collection and processing of samples for mass cytometry analysis.

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

High-Throughput, Multi-Image Cryohistology of Mineralized Tissues

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious projects to disrupt every gene in the mouse genome. The research community needs rapid and inexpensive means to phenotype tissues via histology. Histological analyses of skeletal tissues are often time consuming and semi-quantitative at best, regularly requiring subjective interpretation of slides from trained individuals. Here, we present a cryohistological paradigm for efficient and inexpensive phenotyping of mineralized tissues. First, we present a novel method of tape-stabilized cryosectioning that preserves the morphology of mineralized tissues. These sections are then adhered rigidly to glass slides and imaged repeatedly over several rounds of staining. The resultant images are then aligned either manually or via computer software to yield composite stacks of several layered images. The protocol allows for co-localization of numerous molecular signals to specific cells within a given section. In addition, these fluorescent signals can be quantified objectively via computer software. This protocol overcomes many of the shortcomings associated with histology of mineralized tissues and can serve as a platform for high-throughput, high-content phenotyping of musculoskeletal tissues moving forward.

In this manuscript, we present a high-throughput, semi-automated cryohistology platform to produce aligned composite images of multiple response measures from several rounds of fluorescent imaging on frozen sections of mineralized tissues.

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious ...