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.

<table><tbody><tr><td>1-1/8&quot; plunge cutting carbide for composites</td><td>Warrior</td><td>61812</td><td>28.6mm plunge</td></tr><tr><td>70% Ethanol</td><td>Fisher Scientific</td><td>BP8201500</td><td>3.8 Liters</td></tr><tr><td>Blunt-tipped forceps</td><td>Fisher Scientific</td><td>10-300</td><td></td></tr><tr><td>Centrifuge tubes</td><td>ThermoFisher</td><td>55398</td><td></td></tr><tr><td>Crystalbond 509-3 Epoxy</td><td>Ted Pella</td><td>821-3</td><td></td></tr><tr><td>CTAnalyser</td><td>Bruker microCT</td><td>v.1.15.4.0</td><td>Download and install at https://www.bruker.com/products/microtomography/micro-ct-software/3dsuite.html</td></tr><tr><td>Dental Tool Kit</td><td>Amazon</td><td>787269885110</td><td></td></tr><tr><td>Diamond wafering saw blade for composite material</td><td>Buehler</td><td>#11-4247</td><td></td></tr><tr><td>Drill Press</td><td>Jet Mill/Drill</td><td>350017</td><td>Model: JMD-15, benchtop drill presses are suitable substites, but typically lack a translatable machine table for positioning samples beneath the drill stem</td></tr><tr><td>Fine-tipped forceps</td><td>Fisher Scientific</td><td>22-327379</td><td></td></tr><tr><td>Fixturing clamps for XY machine table for mill/drill</td><td>MSC Industrial Supply</td><td>#04804571</td><td></td></tr><tr><td>Glass microscope slides</td><td>Ted Pella</td><td>26005</td><td>75x50mm slides, 1mm thick</td></tr><tr><td>Glass slide chuck</td><td>Buehler</td><td>#112488</td><td>Large enough to hold 75x50mm glass slides</td></tr><tr><td>Hot plate capable of reaching 140 &amp;deg;C</td><td>ThermoScientific</td><td>HP88850105</td><td></td></tr><tr><td>Incubator</td><td>NAPCO</td><td>Model 4200</td><td></td></tr><tr><td>Isocut Fluid</td><td>Buehler</td><td>111193032</td><td>Lubricant; 30mL</td></tr><tr><td>Jeweler&#039;s diamond coring drill bit</td><td>Otto Frei</td><td>#119.050</td><td>2mm inner diameter hollow stem coring bit</td></tr><tr><td>NRecon</td><td>Bruker microCT</td><td>v.1.6.10.2</td><td>Download and install at https://www.bruker.com/products/microtomography.html</td></tr><tr><td>Oscillating saw</td><td>Harbor Freight</td><td>62866</td><td></td></tr><tr><td>Oven-safe glass dishes</td><td>Pyrex</td><td>1117715</td><td>Glass food storage container</td></tr><tr><td>Precision slow-speed saw (Isomet 1000)</td><td>Buehler</td><td>111280160</td><td></td></tr><tr><td>Razor blades</td><td>Amazon</td><td>25181</td><td></td></tr><tr><td>Shallow aluminum tins</td><td>Amazon</td><td>B01MRWLD0R</td><td>~8cm diameter</td></tr><tr><td>Specimen cups</td><td>Amazon</td><td>616784425436 885334344729</td><td></td></tr><tr><td>Tergazyme detergent</td><td>Alconox</td><td>1304-1</td><td>1.8kg box</td></tr><tr><td>Ultrasonic cleaner</td><td>MTI Corporation</td><td>KJ201508006</td><td></td></tr></tbody></table>

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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Research

JoVE Journal

Biology

5.0K Views.  The University of Akron. We gebruikten een geologisch (coring) bemonsteringsprotocol om corticale botmonsters van uniforme grootte aan te schaffen voor SRμCT-experimenten vanuit het voorste aspect van menselijke femora. Deze methode is minimaal destructief, efficiënt, resulteert in cilindrische specimens die beeldvormingsartefacten uit onregelmatige monstervormen minimaliseren en microarchitecturale visualisatie en analyse verbeteren.

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

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (&mu;CT).
High resolution &mu;CT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the &mu;CT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, &mu;CT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in &mu;CT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180&deg; or 360&deg;between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

Bioengineering

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

Developmental Biology

Preparing Lamellae from Vitreous Biological Samples Using a Dual-Beam Scanning Electron Microscope for Cryo-Electron Tomography

Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected&#160;human erythrocytes, which could easily be adapted for other biological samples. The basic principles for preparing samples, milling, and viewing lamellae are common to all instruments and the protocol can be followed as a general guide to on-grid cryo-lamella preparation for cryo-electron microscopy (cryoEM) and cryo-electron tomography (cryoET). Electron microscopy grids supporting the cells are plunge-frozen into liquid nitrogen-cooled liquid ethane using a manual or automated plunge freezer, then screened on a light microscope equipped with a cryo-stage. Frozen grids are transferred into a cryo-scanning electron microscope equipped with a focused ion beam (cryoFIB-SEM). Grids are routinely sputter coated prior to milling, which aids dispersal of charge build-up during milling. Alternatively, an e-beam rotary coater can be used to apply a layer of carbon-platinum to the grids, the exact thickness of which can be more precisely controlled. Once inside the cryoFIB-SEM an additional coating of an organoplatinum compound is applied to the surface of the grid via a gas injection system (GIS). This layer protects the front edge of the lamella as it is milled, the integrity of which is critical for achieving uniformly thin lamellae. Regions of interest are identified via SEM and milling is carried out in a step-wise fashion, reducing the current of the ion beam as the lamella reaches electron transparency, in order to avoid excessive heat generation. A grid with multiple lamellae is then transferred to a transmission electron microscope (TEM) under cryogenic conditions for tilt-series acquisition. A robust and contamination-free workflow for lamella preparation is an essential step for downstream techniques, including cellular cryoEM, cryoET, and sub-tomogram averaging. Development of these techniques, especially for lift-out and milling of high-pressure frozen samples, is of high-priority in the field.

Using focused ion beam milling to produce vitreous on-grid lamellae from plunge frozen biological samples for cryo-electron tomography.

Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected&#160;human erythrocytes, which could ...

Biochemistry

Outer-Boundary Assisted Segmentation and Quantification of Trabecular Bones by an Imagej Plugin

Micro-computed tomography (micro-CT) is routinely used to assess bone quantity and trabecular microstructural properties in small animals under different bone loss conditions. However, the standard approach for trabecular analysis of micro-CT images is slice-by-slice semi-automatic hand-contouring, which is labor intensive and error prone. Described here is an efficient method for automatic segmentation of trabecular bones according to the bone's outer boundaries, where trabecular bones can be identified and segmented automatically with accuracy with less operator bias when appropriate segmentation parameters are set. To profile satisfactory segmentation parameters, an image stack of segmentation results is displayed, where all possible combinations of the segmentation parameters are changed one by one in sequence, and segmentation results with associated parameters can easily be visually checked. As a quality-control feature of the plugin, simulated standard objects are quantified where the measured quantities can be compared with theoretical values. Layer-by-layer quantification of trabecular properties and trabecular thicknesses are reported by such a plugin, and the distributions of such properties within the selected regions can be profiled easily. Although layer-by-layer quantification retains more information about trabecular bones and facilitates further statistical analysis of structural changes, such measures are unavailable from the output of current commercial software, where only a single quantified value for each parameter is reported for each sample. Therefore, the described workflows are better approaches for analyzing trabecular bones with accuracy and efficiency.

We present a workflow for segmenting and quantifying trabecular bones for 2D and 3D images based on the bone&#39;s outer boundary using an ImageJ plugin. This approach is more efficient and accurate than the current manual hand-contouring approach, and provides layer-by-layer quantifications, which are not available in current commercial software.

Micro-computed tomography (micro-CT) is routinely used to assess bone quantity and trabecular microstructural properties in small animals under different ...

3D Imaging of Soft-Tissue Samples using an X-ray Specific Staining Method and Nanoscopic Computed Tomography

We demonstrate a laboratory-based method combining X-ray microCT and nanoCT with a specific X-ray stain, which targets the cell cytoplasm. The described protocol is easy to apply, fast and suitable for larger soft-tissue samples. The presented methodology enables the characterization of crucial tissue structures in three dimensions and is demonstrated on a whole mouse kidney. The multiscale approach allows to image the entire mouse kidney and supports the selection of further volumes of interest, which are acquired with higher resolutions ranging into the nanometer range. Thereby, soft-tissue morphology with a similar detail level as the corresponding histological light microscopy images is reproduced. Deeper insights into the 3D configuration of tissue structures are achieved without impeding further investigations through histological methods.

A protocol for 3D visualization of microscopic tissue structures by using an X-ray specific staining method designed for X-ray computed tomography is presented.

We demonstrate a laboratory-based method combining X-ray microCT and nanoCT with a specific X-ray stain, which targets the cell cytoplasm. The described ...

A Micro-CT-based Method for Characterizing Lesions and Locating Electrodes in Small Animal Brains

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that requires manual estimation. Here, we describe a simple, straightforward method for quantifying lesions and locating electrodes in the brain that is less laborious and yields more detailed results. Whole brains are stained with osmium tetroxide, embedded in resin, and imaged with a micro-CT scanner. The scans result in 3D digital volumes of the brains with resolutions and virtual section thicknesses dependent on the sample size (12&#8211;15 and 5&#8211;6 &#181;m&#160;per voxel for rat and zebra finch brains, respectively). Surface and deep lesions can be characterized, and single tetrodes, tetrode arrays, electrolytic lesions, and silicon probes can also be localized. Free and proprietary software allows experimenters to examine the sample volume from any plane and segment the volume manually or automatically. Because this method generates whole brain volume, lesions and electrodes can be quantified to a much higher degree than in current methods, which will help standardize comparisons within and across studies.

This article describes a straightforward method to prepare small animal brains for micro-CT imaging, in which lesions can be quantified and electrodes located with high precision in the context of the whole brain.

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that ...

Neuroscience

An Unbiased Approach of Sampling TEM Sections in Neuroscience

Investigations of the ultrastructural features of neurons and their synapses are only possible with electron microscopy. Especially for comparative studies of the changes in densities and distributions of such features, an unbiased sampling protocol is vital for reliable results. Here, we present a workflow for the image acquisition of brain samples. The workflow allows systematic uniform random sampling within a defined brain region, and the images can be analyzed using a disector. This technique is much faster than extensive examination of serial sections but still presents a feasible approach to estimate the densities and distributions of ultrastructure features. Before embedding, stained vibratome sections were used as a reference to identify the brain region under investigation, which helped speed up the overall specimen preparation process. This approach was used for comparative studies investigating the effect of an enriched-housing environment on several ultrastructural parameters in the mouse brain. Based on the successful use of the workflow, we adapted it for the purpose of elemental analysis of brain samples. We optimized the protocol in terms of the time of user-interaction. Automating all the time-consuming steps by compiling a script for the open source software SerialEM helps the user to focus on the main work of acquiring the elemental maps. As in the original workflow, we paid attention to the unbiased sampling approach to guarantee reliable results.

We introduce a novel workflow for electron microscopy investigations of brain tissue. The method allows the user to examine neuronal features in an unbiased fashion. For elemental analysis, we also present a script that automatizes most of the workflow for randomized sampling.

Investigations of the ultrastructural features of neurons and their synapses are only possible with electron microscopy. Especially for comparative ...

Principles and Protocols of Mechanical Testing for Bone Fracture Susceptibility

Practical Considerations for the Design, Execution, and Interpretation of Studies Involving Whole-Bone Bending Tests of Rodent Bones

Skeletal fragility leading to fracture is an American public health crisis resulting in 1.5 million fractures each year and $18 billion in direct care costs. The ability to understand the mechanisms underlying bone disease and the response to treatment is not only desired, but critical. Mechanical testing of bone serves as a valuable technique for understanding and quantifying a bone&#39;s susceptibility to fracture. While this method appears simple to perform, inappropriate and inaccurate conclusions may be reached if governing assumptions and key steps are disregarded by the user. This has been observed across disciplines as studies continue to be published with misuse of methods and incorrect interpretation of results. This protocol will serve as a primer for the principles associated with mechanical testing along with the application of these techniques-from considerations of sample size through tissue harvesting and storage, to data analysis and interpretation. With this in hand, valuable information regarding a bone&#39;s susceptibility to fracture may be obtained, furthering understanding for both academic research and clinical solutions.

Author Spotlight: Enhancing Accuracy and Reproducibility in Whole Bone Bending Tests 

Mechanical testing of rodent bones is a valuable method for extracting information regarding a bone's susceptibility to fracture. Lacking proper practical understanding, the results may be overinterpreted or lack validity. This protocol will serve as a guide to ensure mechanical tests are performed accurately to provide valid and functional data.

Skeletal fragility leading to fracture is an American public health crisis resulting in 1.5 million fractures each year and $18 billion in direct care ...

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 ...

HE Staining and Micro-CT to Evaluate Murine Trabecular Bone Microarchitecture

Trabecular Bone Microarchitecture Evaluation in an Osteoporosis Mouse Model

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton is crucial for gaining insight into the pathophysiology of osteoporosis and improving its treatment. However, handling bone samples can be complex due to their hard and dense properties. Secondly, specialized software makes image processing and analysis difficult. In this protocol, we present a cost-effective and easy-to-use solution for trabecular bone microstructure analysis. Detailed steps and precautions are provided. Micro-CT is a non-destructive three-dimensional (3D) imaging technique that provides high-resolution images of trabecular bone structure. It allows for the objective and quantitative evaluation of bone quality, which is why it is widely regarded as the gold standard method for bone quality assessment. However, histomorphometry remains indispensable as it offers crucial cellular-level parameters, bridging the gap between two-dimensional (2D) and 3D assessments of bone specimens. As for the histologic techniques, we chose to decalcify the bone tissue and then perform traditional paraffin embedding. In summary, combining these two methods can provide more comprehensive and accurate information on bone microstructure.

Author Spotlight: An Economic and Efficient Method for Quantitative Evaluation of Bone Microarchitecture in a Murine Osteoporosis Model 

This protocol presents an economical and efficient method for quantitative evaluation of bone microarchitecture in a mouse model of osteoporosis by combining Hematoxylin-Eosin (HE) staining and micro-computed tomography (Micro-CT) techniques.

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton ...

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

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