Name: a sectioning, coring, and image processing guide for high-throughput cortical bone sample procurement and analysis for synchrotron micro-ct
Uploaded: 2020-06-12T14:00:00
Description: 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

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 availab...

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W., 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=Beamlines+of+the+biomedical+imaging+and+therapy+facility+at+the+Canadian+light+source+-+part+3.">Beamlines of the biomedical imaging and therapy facility at the Canadian light source - part 3.</a> Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment. 775, 1-4 (2015).</li><li>Carter, Y., Suchorab, J. L., Thomas, C. D. L., Clement, J. G., Cooper, D. M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+variation+in+cortical+osteocyte+lacunar+parameters+in+healthy+young+males.">Normal variation in cortical osteocyte lacunar parameters in healthy young males.</a> Journal of Anatomy. 225 (3), 328-336 (2014).</li><li>Carter, Y., Thomas, C. D. L., Clement, J. G., Cooper, D. 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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 border="0" cellpadding="0" cellspacing="0" style="width:1367px;" width="1367">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">1-1/8&#34; plunge cutting carbide for composites</td>
			<td style="width:120px;">Warrior</td>
			<td style="width:133px;">61812</td>
			<td style="width:899px;">28.6mm plunge</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:215px;">70% Ethanol</td>
			<td style="width:120px;">Fisher Scientific</td>
			<td style="width:133px;">BP8201500</td>
			<td>3.8 Liters</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Blunt-tipped forceps</td>
			<td style="width:120px;">Fisher Scientific</td>
			<td style="width:133px;">10-300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Centrifuge tubes</td>
			<td style="width:120px;">ThermoFisher</td>
			<td style="width:133px;">55398</td>
			<td></td>
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			<td height="21" style="height:21px;width:215px;">Crystalbond 509-3 Epoxy</td>
			<td style="width:120px;">Ted Pella</td>
			<td style="width:133px;">821-3</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:215px;">CTAnalyser</td>
			<td style="width:120px;">Bruker microCT</td>
			<td style="width:133px;">v.1.15.4.0</td>
			<td>Download and install at https://www.bruker.com/products/microtomography/micro-ct-software/3dsuite.html</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Dental Tool Kit</td>
			<td style="width:120px;">Amazon</td>
			<td style="width:133px;">787269885110</td>
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			<td height="42" style="height:42px;width:215px;">Diamond wafering saw blade for composite material</td>
			<td style="width:120px;">Buehler</td>
			<td style="width:133px;">#11-4247</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Drill Press</td>
			<td style="width:120px;">Jet Mill/Drill</td>
			<td style="width:133px;">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 height="21">
			<td height="21" style="height:21px;width:215px;">Fine-tipped forceps</td>
			<td style="width:120px;">Fisher Scientific</td>
			<td style="width:133px;">22-327379</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Fixturing clamps for XY machine table for mill/drill</td>
			<td style="width:120px;">MSC Industrial Supply</td>
			<td style="width:133px;">#04804571</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Glass microscope slides</td>
			<td style="width:120px;">Ted Pella</td>
			<td style="width:133px;">26005</td>
			<td>75x50mm slides, 1mm thick</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Glass slide chuck</td>
			<td style="width:120px;">Buehler</td>
			<td style="width:133px;">#112488</td>
			<td>Large enough to hold 75x50mm glass slides</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Hot plate capable of reaching 140 &#176;C</td>
			<td style="width:120px;">ThermoScientific</td>
			<td style="width:133px;">HP88850105</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Incubator</td>
			<td style="width:120px;">NAPCO</td>
			<td>Model 4200</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Isocut Fluid</td>
			<td style="width:120px;">Buehler</td>
			<td style="width:133px;">111193032</td>
			<td>Lubricant; 30mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Jeweler&#39;s diamond coring drill bit</td>
			<td style="width:120px;">Otto Frei</td>
			<td style="width:133px;">#119.050</td>
			<td>2mm inner diameter hollow stem coring bit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">NRecon</td>
			<td style="width:120px;">Bruker microCT</td>
			<td style="width:133px;">v.1.6.10.2</td>
			<td>Download and install at https://www.bruker.com/products/microtomography.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Oscillating saw</td>
			<td style="width:120px;">Harbor Freight</td>
			<td style="width:133px;">62866</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Oven-safe glass dishes</td>
			<td style="width:120px;">Pyrex</td>
			<td style="width:133px;">1117715</td>
			<td>Glass food storage container</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Precision slow-speed saw (Isomet 1000)</td>
			<td style="width:120px;">Buehler</td>
			<td style="width:133px;">111280160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Razor blades</td>
			<td style="width:120px;">Amazon</td>
			<td style="width:133px;">25181</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Shallow aluminum tins</td>
			<td style="width:120px;">Amazon</td>
			<td style="width:133px;">B01MRWLD0R</td>
			<td>~8cm diameter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Specimen cups</td>
			<td style="width:120px;">Amazon</td>
			<td style="width:133px;">616784425436 885334344729</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Tergazyme detergent</td>
			<td style="width:120px;">Alconox</td>
			<td style="width:133px;">1304-1</td>
			<td>1.8kg box</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Ultrasonic cleaner</td>
			<td style="width:120px;">MTI Corporation</td>
			<td style="width:133px;">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 ...

Available data related to bone specimen procurement for synchrotron micro-computed tomography remain sparse. Our comprehensive tutorial provides a methodology that is straightforward, minimally destructive, and cost-effective. Procuring bone specimens with consistent dimensions and a cylindrical shape is critical to ensure that the resulting data sets are of the highest quality and the results are applicable.The techniques described in this manuscript are applicable to coring rocks, fossils, or any hard material. We've used these to collect uniformly sized cores of rocks and single crystals for high-pressure experiments to explore the rheology of Earth's crust and upper mantle. Novice corers may find that the bone specimen forms conical shapes.This can be addressed by allowing adequate and enough time for bone dust to flush from the drill bit and slowing down coring speed. There's a lack of instructional texts and videos that demonstrate bone specimen procurement for synchrotron micro-computed tomography and is the logical analysis. Our step-by-step bone preparation tutorial helps to fill this out gap.Place a 75 by 25 millimeter glass microscope slide on hot plates up to 140 degrees Celsius and melt a generous amount of thermal epoxy resin on the center of the slide. 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.Let the mounted specimen rest on the hot plate for approximately five minutes to allow the thermal epoxy to wick into the pores and cracks making sure that the epoxy on the slide is free of bubbles. If bubbles are present, shift the sample back and forth to remove them. Use blunt forceps to remove the slide with the mounted specimen from the hot plate and allow it to cool at room temperature for about 10 minutes, then remove any epoxy from the edge of the slide with a razor blade to ensure that the chuck adequately grips the slide.Attach the slide with the adhered sample to a glass slide chuck and mount the chuck on the swivel arm of a slow speed sectioning saw positioning it so that a cross-section of the bone can be cut perpendicular to its length. Adjust the swivel arm to ensure that the blade contacts and transects the sample. Add weights to the far side of the cutting arm to counter the weight of the arm and add distilled water and cutting fluid to the fluid receptacle.Tightly secure the diamond wafer blade and ensure that the fluid level submerges the cutting portion of the blade. Set the speed to 200 RPM and slowly lower the sample onto the blade. Once the saw begins sectioning, ensure that the blade and chuck are not wobbling or bouncing.If they are, immediately stop the saw and tighten the blade or chuck arm assembly. If the chuck is aggressively moving up and down, add more counterweights. The first thick section is a waste cut that will provide a well-defined surface parallel to each additional cut.After the initial waste cut, raise the swivel arm and move the chuck five millimeters towards the blade using the positioning dial. After sectioning is complete, place the glass slide with the mounted specimen on a hot plate to melt the thermal epoxy. Mount five millimeter bone sections to the bottom of a shallow aluminum tin using the thermal epoxy bonding technique previously described.Place the tin on an XY machine table of the mill drill press and hand-tighten the fixturing clamps. Insert a two millimeter inner diameter hollow shaft jewelers diamond tipped coring drill bit into the mill drill chuck and adjust the depth limiter to prevent coring through the tin. Align the central anterior aspect of the bone sample beneath the drill bit while avoiding close contact with the periosteum and ostium or highly trabecularized areas.Fill the tin with distilled water to completely cover the sample which prevents heat buildup, burning of the sample, and damage to the drill bit during coring. The mill drill press can be dangerous if proper safety measures aren't taken. Operators should ensure that they wear safety glasses, that loose clothing is secured, and that long hair is pulled back so it doesn't get caught in the spindle.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. During coring, lift the drill bit in and out of the sample while keeping the bit's tip beneath the water surface.Do this every few seconds to flush out trapped bone dust and ensure debris is not occluding the drill bit. After coring is complete, the resulting bone core may become lodged in the hollow stemmed drill bit. Use a pair of fine tipped forceps or a small Allen wrench to dislodge the core from the bit.Store the cored sample in a labeled microcentrifuge tube in a cool and dry location until imaging. The described method of core sampling proved to be highly effective and efficient. Representative figures comparing the image processing workflow of a cored sample and one procured using a rotary tool are shown here.The sample cut using the common rotary tool exhibited an increased number of canals and lacunae and a decreased average canal diameter, canal volume, and cortical porosity when compared to the cored sample. This table shows porosity data for each sample. Although the coring protocol decreases artifacts observed in synchrotron microcomputer tomography scans, the lower quality artifact-laden figures from the rectilinear bone block experiments represent a multifaceted issue.Subsequent image processing confirmed the potential of the technique to improve visualization of cortical bone micro architecture. For example, mineralization differences, improved delineation of osteonal boundaries, and consistent visualization of soft tissues within vascular canals were observed. Proceeding slowly is the key to getting consistently-sized cylindrical samples.Going too fast can cause the specimens to become cone-shaped instead of cylindrical. While procuring thick sections for coring, further thick sections can be gathered for Brightfield or confocal microscopy. This allows for the visualization of the canalicular network.

a-sectioning-coring-image-processing-guide-for-high-throughput

Research

JoVE Journal

Biology

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 ...

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 ...

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 ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

Assessment of Global Ocular Structure Following Spaceflight Using a Micro-Computed Tomography (Micro-CT) Imaging Method

Reports show that prolonged exposure to a spaceflight environment produces morphologic and functional ophthalmic changes in astronauts during and after an International Space Station (ISS) mission. However, the underlying mechanisms of these spaceflight-induced changes are currently unknown. The purpose of the present study was to determine the impact of the spaceflight environment on ocular structures by evaluating the thickness of the mouse retina, the retinal pigment epithelium (RPE), the choroid and the sclera layer using micro-CT imaging. Ten-week-old C57BL/6 male mice were housed aboard the ISS for a 35-day mission and then returned to Earth alive for tissue analysis. For comparison, ground control (GC) mice on Earth were maintained in identical environmental conditions and hardware. Ocular tissue samples were collected for micro-CT analysis within 38(&#177;4) hours after splashdown. The images of the cross-section of the retina, the RPE, the choroid, and the sclera layer of the fixed eye was recorded in an axial and sagittal view using a micro-CT imaging acquisition method. The micro-CT analysis showed that the cross-section areas of the retina, RPE, and choroid layer thickness were changed in spaceflight samples compared to GC, with spaceflight samples showing significantly thinner cross-sections and layers compared to controls. The findings from this study indicate that micro-CT evaluation is a sensitive and reliable method to characterize ocular structure changes. These results are expected to improve the understanding of the impact of environmental stress on global ocular structures.

Assessment of Global Ocular Structure Following Spaceflight Using a Micro-Computed Tomography Micro-CT Imaging Method

We present a protocol using high-resolution micro-computed tomography imaging to determine whether spaceflight induced damage on ocular structures. The protocol shows the micro-CT-derived measurement of ex vivo rodent ocular structures. We demonstrate the ability to assess ocular morphological changes following spaceflight using a non-destructive tridimensional technique to evaluate ocular damage.

Reports show that prolonged exposure to a spaceflight environment produces morphologic and functional ophthalmic changes in astronauts during and after an ...

A High-Throughput Platform for Culture and 3D Imaging of Organoids

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by standard imaging approaches. This protocol describes a way to prepare microfabricated organoid culture chips, which enable multiscale, 3D live imaging on a user-friendly instrument requiring minimal manipulations and capable of up to 300 organoids/h imaging throughput. These culture chips are compatible with both air and immersion objectives (air, water, oil, and silicone) and a wide range of common microscopes (e.g., spinning disk, point scanner confocal, wide field, and brightfield). Moreover, they can be used with light-sheet modalities such as the single-objective, single-plane illumination microscopy (SPIM) technology (soSPIM). 
The protocol described here gives detailed steps for the preparation of the microfabricated culture chips and the culture and staining of organoids. Only a short length of time is required to become familiar with, and consumables and equipment can be easily found in normal biolabs. Here, the 3D imaging capabilities will be demonstrated only with commercial standard microscopes (e.g., spinning disk for 3D reconstruction and wide field microscopy for routine monitoring).

This paper presents a fabrication protocol for a new type of culture substrate with hundreds of microcontainers per mm2, in which organoids can be cultured and observed using high-resolution microscopy. The cell seeding and immunostaining protocols are also detailed.

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by ...

Elucidating Glycan Structure and Occurrence Using Microarray Polymer Profiling

Microarray Polymer Profiling (MAPP) for High-Throughput Glycan Analysis

Microarray polymer profiling (MAPP) is a robust and reproducible approach to systematically determine the composition and relative abundance of glycans and glycoconjugates within a variety of biological samples, including plant and algal tissues, food materials, and human, animal, and microbial samples. Microarray technology underpins the efficacy of this method by providing a miniaturized, high-throughput screening platform, allowing thousands of interactions between glycans and highly specific glycan-directed molecular probes to be characterized concomitantly, using only small amounts of analytes. Constituent glycans are chemically and enzymatically fractionated, before being sequentially extracted from the sample and directly immobilized onto nitrocellulose membranes. The glycan composition is determined by the attachment of specific glycan-recognizing molecular probes to the extorted and printed molecules. MAPP is complementary to conventional glycan analysis techniques, such as monosaccharide and linkage analysis and mass spectrometry. However, glycan-recognizing molecular probes provide insight into the structural configurations of glycans, which can aid in elucidating biological interactions and functional roles.

Author Spotlight: MAPP Protocol &amp;#8211; Advancing Glycan Analysis 

Microarray polymer profiling (MAPP) is a high-throughput technique for compositional analysis of glycans in biological samples.

Microarray polymer profiling (MAPP) is a robust and reproducible approach to systematically determine the composition and relative abundance of glycans ...