This study was supported by a faculty research grant of Yonsei University College of Medicine (6-2018-0099). The authors thank the people who very generously donated their bodies to Yonsei University College of Medicine. We are grateful to Jun Ho Kim and Jong Ho Bang for their technical support (staff members in the Surgical Anatomy Education Centre at the Yonsei University College of Medicine). We are also grateful to Genoss Co., Ltd. for the high-quality microCT scanning system used in this research.

All cadavers utilized in this study were legally donated to the Surgical Anatomy Education Centre at Yonsei University College of Medicine.
1. Obtaining samples
<ol>
	<li>Draw an incision line on the cadaver with a colored pencil to indicate the cutting area for sample harvesting. Check that the incision line drawn extends medially to a medial canthus, laterally to a lateral canthus, superiorly to a superior border of the lower eyelid, and inferiorly to 1 cm below the line from the orbital rim. 
	NOTE: Consider the sample size based on the maximum scanning size of the micro-CT equipment (our equipment could acquire an image with a maximum object dimension of 7 &#215; 7 cm). Here, a sample approximately 1 cm in width, 3 cm in length, and 1.25 g weight was harvested from the ORL region.</li>
	<li>Cut the facial tissues following the incision line with a blade. Make sure the cut is deep such that the tip of the blade touches the bone. The sample must include the skin, subcutaneous tissue, muscle, fat, and periosteum.</li>
	<li>Fix the sample in 10% formalin immediately and preserve it for 5 to 7 days at room temperature (Figure 1A). 
	NOTE: Both embalmed and fresh cadavers can be used for this study. However, the fixation solution for cadavers might differ slightly from the solution used in a biological experiment. Therefore, we suggest fixing the sample with 10% formalin again even after obtaining the sample from embalmed cadavers.</li>
</ol>
2. Preparation for staining
<ol>
	<li>After fixing, slice the sample into 3 pieces (5&#8211;7 mm in thickness). Do not lose the location and direction of each piece during this process. 
	NOTE: The microCT scanner we use can cover a maximum size of 7 cm&#179;, but the PTA solution cannot penetrate the sample successfully if it is too thick.</li>
	<li>Sew the superolateral side of each piece using a needle and black thread such that the direction of the sample can be checked later.</li>
	<li>Dehydrate the sample in a series of 30%, 50%, and 70% ethanol solutions for 1 day each.</li>
	<li>Place the sample in 70% ethanol until staining.</li>
</ol>
3. PTA preparation
<ol>
	<li>Begin the PTA staining process 1 week before microCT scanning is scheduled.</li>
	<li>Prepare 210&#160;mL of 70% ethanol solution and add 2.1&#160;g of PTA power to it. Mix well using a shaker at 55-60 rpm. 
	NOTE: The concentration of the PTA solution should be 1% in ethanol.</li>
	<li>Prepare three&#160;70 mL plastic containers&#160;for each sliced piece. Fill the containers with the PTA solution. Soak the specimens into the containers and place them on a shaker for effective penetration. Leave the samples for 5&#8211;7 days (Figure 1B).</li>
	<li>When the staining is completed, store the sample in 70% ethanol to prepare for scanning. 
	NOTE: The stained samples can be maintained for several months, but it is recommended that the samples be scanned as soon as possible to ensure full staining.</li>
</ol>
4. MicroCT scanning
<ol>
	<li>Wrap the sample with parafilm to prevent drying. Do not wrap the samples too tight, as that may lead to deformation.</li>
	<li>Open the scanner and place the sample on the tray (Figure 2).</li>
	<li>Set the scanning parameters as follows: source voltage (kV) = 70, source current (&#956;A) = 114, Al filter = 0.5 mm, image pixel size (&#956;m&#178;) = 20, pixels = 2240 &#215; 2240, exposure (ms) = 500, rotation step (deg) = 0.3. 
	NOTE: The parameters may be modified according to the samples and/or scanners used.</li>
	<li>Start scanning. 
	NOTE: Scanning takes 30 to 60 min depending on the intended resolution and the speed of the scanner.</li>
</ol>
5. Reconstruction and optimization of data
<ol>
	<li>Run the reconstruction software. Select Open Dataset on the Actions menu to launch the scanned files.</li>
	<li>Select the Settings tab on the Reconstruction window. Set the parameters as follows: Ring artifacts reduction = 7, Beam-hardening correction (%) = 40. 
	NOTE: The parameters can be modified according to the sample.</li>
	<li>Begin reconstruction by selecting Start on the Start tab.&#160;The final data will be stored in the designated folder.</li>
	<li>Run the file resizing software. Select Source data set to launch the reconstructed files.</li>
	<li>Select jpg on the Destination data set tab.</li>
	<li>Choose the Resizing option 1/2 with a Quality option of No interpolation (fast).</li>
	<li>Adjust the slide bar to 100 (highest) in the Image compression tab. Start converting. 
	NOTE: The resizing option is to avoid slowing down the computer speed when 3D rendered; however, it can result in lower resolution when resized extensively. We suggest resizing in half for acceptable resolution with better handling.</li>
</ol>
6. 3D reconstruction
<ol>
	<li>Run the 3D volume rendering software.</li>
	<li>Select Actions &#62; Load volume data to launch the dataset.</li>
	<li>Adjust the brightness and contrast level by modifying shape transfer function in histogram in the Transfer Function Editor tab.</li>
	<li>Select Options &#62; Lighting.</li>
	<li>Select Shadows and Surface Lighting icons. These effects provide a realistic modeling tone.</li>
	<li>Find the best view by moving (click and drag), rotating (right-click and drag), and zooming in or out of (scroll) the model.</li>
	<li>Slide the plane (shift + click and drag in the inner direction) to show the sectional images (Figure 3).</li>
	<li>Turn on Light icon. Adjust the lighting indication bar and find the best lighting for viewing. Then, turn off the icon and close the Lighting tab.</li>
	<li>Select Options &#62; Show &#62; Clipping Box to hide the box for the final image.</li>
	<li>Select Actions &#62; Save image to store the image.</li>
</ol>

<ol><li>Nierenberger, M., Remond, Y., Ahzi, S., Choquet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessing+the+three-dimensional+collagen+network+in+soft+tissues+using+contrast+agents+and+high+resolution+micro-CT%3A+Application+to+porcine+iliac+veins%5BTitle%5D)+AND+%22Comptes+Rendus+Biologies%22%5BJournal%5D)">Assessing the three-dimensional collagen network in soft tissues using contrast agents and high resolution micro-CT: Application to porcine iliac veins.</a> Comptes Rendus Biologies. 338 (7), 425-433 (2015).</li>
<li>Vymazalová, K., Vargová, L., Zikmund, T., Kaiser, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+possibilities+of+studying+human+embryos+and+foetuses+using+micro-CT%3A+a+technical+note%5BTitle%5D)+AND+%22Anatomical+Science+International%22%5BJournal%5D)">The possibilities of studying human embryos and foetuses using micro-CT: a technical note.</a> Anatomical Science International. 92 (2), 299-303 (2017).</li>
<li>Tesařová, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+micro+computed-tomography+and+3D+printing+for+reverse+engineering+of+mouse+embryo+nasal+capsule%5BTitle%5D)+AND+%22Journal+of+Instrumentation%22%5BJournal%5D)">Use of micro computed-tomography and 3D printing for reverse engineering of mouse embryo nasal capsule.</a> Journal of Instrumentation. 11 (3), 1-11 (2016).</li>
<li>Nemetschek, T., Riedl, H., Jonak, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Topochemistry+of+the+binding+of+phosphotungstic+acid+to+collagen%5BTitle%5D)+AND+%22Journal+of+Molecular+Biology%22%5BJournal%5D)">Topochemistry of the binding of phosphotungstic acid to collagen.</a> Journal of Molecular Biology. 133 (1), 67-83 (1979).</li>
<li>Rao, R. N., Fallman, P. M., Falls, D. G., Meloan, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+comparative+study+of+PAS-phosphotungstic+acid-Diamine+Supra+Blue+FGL+and+immunological+reactions+for+type+I+collagen%5BTitle%5D)+AND+%22Histochemistry%22%5BJournal%5D)">A comparative study of PAS-phosphotungstic acid-Diamine Supra Blue FGL and immunological reactions for type I collagen.</a> Histochemistry. 91 (4), 283-289 (1989).</li>
<li>Metscher, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MicroCT+for+comparative+morphology%3A+simple+staining+methods+allow+high-contrast+3D+imaging+of+diverse+non-mineralized+animal+tissues%5BTitle%5D)+AND+%22BMC+Physiology%22%5BJournal%5D)">MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues.</a> BMC Physiology. 9 (11), (2009).</li>
<li>Metscher, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MicroCT+for+Developmental+Biology%3A+A+Versatile+Tool+for+High-Contrast+3D+Imaging+at+Histological+Resolutions%5BTitle%5D)+AND+%22Developmental+Dynamics%22%5BJournal%5D)">MicroCT for Developmental Biology: A Versatile Tool for High-Contrast 3D Imaging at Histological Resolutions.</a> Developmental Dynamics. 238 (3), 632-640 (2009).</li>
<li>Nieminen, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Determining+collagen+distribution+in+articular+cartilage+using+contrastenhanced+micro-computed+tomography%5BTitle%5D)+AND+%22Osteoarthritis+Cartilage%22%5BJournal%5D)">Determining collagen distribution in articular cartilage using contrastenhanced micro-computed tomography.</a> Osteoarthritis Cartilage. 23 (9), 1613-1621 (2015).</li>
<li>Kwon, O. J., Kwon, H., Choi, Y., Cho, T., Yang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three-dimensional+structure+of+the+orbicularis+retaining+ligament%3A+an+anatomical+study+using+micro+computed+tomography%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Three-dimensional structure of the orbicularis retaining ligament: an anatomical study using micro computed tomography.</a> Scientific Reports. 8 (1), 17042(2018).</li>
<li>Dullin, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%CE%BCCT+of+ex-vivo+stained+mouse+hearts+and+embryos+enables+a+precise+match+between+3D+virtual+histology%2C+classical+histology+and+immunochemistry%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">μCT of ex-vivo stained mouse hearts and embryos enables a precise match between 3D virtual histology, classical histology and immunochemistry.</a> PLoS One. 12 (2), e0170597(2017).</li>
<li>Zikmund, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-contrast+differentiation+resolution+3D+imaging+of+rodent+brain+by+X-ray+computed+microtomography%5BTitle%5D)+AND+%22Journal+of+Instrumentation%22%5BJournal%5D)">High-contrast differentiation resolution 3D imaging of rodent brain by X-ray computed microtomography.</a> Journal of Instrumentation. 13 (2), 1-12 (2018).</li>
<li>Anderson, R., Maga, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+procedure+for+rapid+imaging+of+adult+mouse+brains+with+MicroCT+using+iodine-based+contrast%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">A novel procedure for rapid imaging of adult mouse brains with MicroCT using iodine-based contrast.</a> PLoS One. 10 (11), e0142974(2015).</li>
<li>Nieminen, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((3D+histopathological+grading+of+osteochondral+tissue+using+contrast-enhanced+micro-computed+tomography%5BTitle%5D)+AND+%22Osteoarthritis+Cartilage%22%5BJournal%5D)">3D histopathological grading of osteochondral tissue using contrast-enhanced micro-computed tomography.</a> Osteoarthritis Cartilage. 26 (8), 1118-1126 (2018).</li>
<li>Greef, D. D., Buytaert, J. A. N., Aerts, J. R. M., Hoorebeke, L. V., Dierick, M., Dirckx, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Details+of+Human+Middle+Ear+Morphology+Based+on+Micro-CT+Imaging+of+Phosphotungstic+Acid+Stained+Samples%5BTitle%5D)+AND+%22Journal+of+Morphology%22%5BJournal%5D)">Details of Human Middle Ear Morphology Based on Micro-CT Imaging of Phosphotungstic Acid Stained Samples.</a> Journal of Morphology. 276 (9), 1025-1046 (2015).</li>
<li>Sutter, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Contrast-Enhanced+Microtomographic+Characterisation+of+Vessels+in+Native+Bone+and+Engineered+Vascularised+Grafts+Using+Ink-Gelatin+Perfusion+and+Phosphotungstic+Acid%5BTitle%5D)+AND+%22Contrast+Media+%26+Molecular+Imaging%22%5BJournal%5D)">Contrast-Enhanced Microtomographic Characterisation of Vessels in Native Bone and Engineered Vascularised Grafts Using Ink-Gelatin Perfusion and Phosphotungstic Acid.</a> Contrast Media & Molecular Imaging. 2017, (2017).</li>
</ol>

The authors have nothing to disclose.

We implemented microCT with PTA preparation in the examination of human soft tissues. Briefly, specimens are harvested and fixed in formalin for a few days, followed by dehydration in serial ethanol solutions. Placing the sample into the PTA solution directly after formalin fixation can result in some tissue cracking due to rapid dehydration. Therefore, serial dehydration is needed before PTA staining. Next, the samples are stained using PTA solution for about a week. MicroCT scanning, 3D reconstruction, and analysis can...

Manual dissection and histological observation are typically used to examine human tissues, such as muscles and connective tissues. However, manual dissection can easily damage delicate structures, and histological observation provides limited information about flat cross-sectional surfaces1,2. Therefore, improved methods are needed to examine tissues more precisely and efficiently.
Conventional computed tomography (CT) is generally used in clinical practice, but it lacks the ability to distinguish small structures2,3. Micro X-ray CT (microCT) is an effective tool for obtaining three-dimensional (3D) information of small structures from specimens, without destroying them. However, microCT has limited applications because only dense tissues can be visualized clearly; it cannot be used to differentiate soft tissues. To overcome this limitation, staining agents can be used. Contrast-enhancing agents, like phosphotungstic acid (PTA), phosphomolybdic acid, and Lugol&#8217;s iodine, improve the soft tissue contrast rate during scanning4,5. Several studies comparing these agents suggest that PTA demonstrates good performance and is easy to handle6,7,8.
The orbicularis retaining ligament (ORL) is a delicate structure around the orbit, which can be easily damaged during conventional observation9. We examined and successfully retrieved 3D information on this structure using microCT with PTA as a contrast agent. This method can be applied to studies on other human tissues, such as the heart and liver, with appropriate modifications10,11,12.

<table><tbody><tr><td>12 Tungsto(VI)phosphoric acid n-hydrate&lt;br/&gt; Phosphotungstic acid</td><td>Junsei</td><td>84220-0410</td><td>PTA powder</td></tr><tr><td>CTvox</td><td>Bruker</td><td>ver 2.7</td><td>3D recon software</td></tr><tr><td>Nrecon</td><td>Bruker</td><td>ver 1.7.0.4</td><td>Reconstruction software</td></tr><tr><td>Skyscan</td><td>Bruker</td><td>1173</td><td>MicroCT scanner</td></tr><tr><td>Tconv</td><td>Bruker</td><td>ver 2.0</td><td>File resizing software</td></tr></tbody></table>

use of micro x-ray computed tomography with phosphotungstic acid preparation to visualize human fibromuscular tissue

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate structures&#160;while processing and histological observation provide limited information through cross-sectional imaging. Micro X-ray computed tomography (microCT) is an effective tool for obtaining three-dimensional information without damaging specimens. However, it shows limited efficiency in differentiating soft tissue parts. Use of contrast-enhancing agents, like phosphotungstic acid (PTA), can solve this problem by improving soft tissue contrast. We implemented microCT with PTA to investigate the human orbicularis retaining ligament (ORL), which is a delicate structure in the orbit area. In this method, harvested specimens are fixed in formalin, dehydrated in serial ethanol solutions, and stained with a PTA solution. After staining, microCT scanning, 3D reconstruction, and analysis are performed. Skin, ligaments, and muscles can be clearly visualized using this method. The specimen size and duration of staining are essential features of the method. The suitable specimen thickness was about 5&#8211;7 mm, above which the process was slowed, and the optimum duration was 5&#8211;7 days, below which an empty hole in the central area occasionally occurred. To maintain the location and direction of small pieces during cutting, sewing on the same region of each part is recommended. Furthermore, preliminary analyses of the anatomical structure are needed to correctly identify each piece. Parafilm can be used to prevent drying, but care should be taken to prevent specimen distortion. Our multidirectional observation showed that the ORL is composed of a multilayered meshwork of continuous plates, rather than thread-like fibers, as reported previously. These results suggest that microCT scanning with PTA is useful for examining specific compartments within complex structures of human tissue. It may be helpful in the analyses of cancer tissues, nerve tissues, and various organs, like the heart and liver.

The detailed reconstruction of the ORL was achieved by microCT with PTA preparation (Figure 4). The ligamentous fibromuscular structure extending obliquely between the dermis and periosteum was distinctly observed (Figure 4A). In the coronal view (Figure 4B), the amount and complexity of fibers increased laterally. In the horizontal view (Figure 4C), an elaborate meshwork with an arborized formation was...

Watch this Scientific Journal Video about Use of Micro X-ray Computed Tomography with Phosphotungstic Acid Preparation to Visualize Human Fibromuscular Tissue at JoVE.com

Use of Micro X-ray Computed Tomography with Phosphotungstic Acid Preparation to Visualize Human Fibromuscular Tissue

Micro X-ray computed tomography is effective in obtaining three-dimensional information from undamaged human specimens but has limited success in observing soft tissues. The use of phosphotungstic acid contrast agent can resolve this issue. We implemented this contrast agent to examine human delicate fibromuscular tissues (the orbicularis retaining ligament).

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate ...

use-micro-x-ray-computed-tomography-with-phosphotungstic-acid

Research

JoVE Journal

Bioengineering

6.7K Views.  Yonsei University College of Medicine. We implementeren microCT met fosfootungstic acid om het contrast van het zachte weefsel te verbeteren in het niet-invasieve onderzoek naar de menselijke orbiculaire orbiculaire ligament, een delicate structuur binnen het baangebied. Dit protocol is geoptimaliseerd om onderzoekers te helpen bij het niet-invasieve onderzoek van menselijke zachte weefsels en is eenvoudig uit te voeren met een redelijke hoeveelheid kosten. Om de monsters te verkrijgen, gebruik eerst een gekleurd potlood om het sn...

Video: Use of Micro X-ray Computed Tomography with Phosphotungstic Acid Preparation to Visualize Human Fibromuscular Tissue

Microfocus X-ray CT (microCT) Imaging of Actinia equina (Cnidaria), Harmothoe sp. (Annelida), and Xenoturbella japonica (Xenacoelomorpha)

Traditionally, biologists have had to rely on destructive methods such as sectioning in order to investigate the internal structures of opaque organisms. Non-destructive microfocus X-ray computed tomography (microCT) imaging has become a powerful and emerging protocol in biology, due to technological advancements in sample staining methods and innovations in microCT hardware, processing computers, and data analysis software. However, this protocol is not commonly used, as it is in the medical and industrial fields. One of the reasons for this limited use is the lack of a simple and comprehensible manual that covers all of the necessary steps: sample collection, fixation, staining, mounting, scanning, and data analyses. Another reason is the vast diversity of metazoans, particularly marine invertebrates. Because of marine invertebrates&#8217; diverse sizes, morphologies, and physiologies, it is crucial to adjust experimental conditions and hardware configurations at each step, depending on the sample. Here, microCT imaging methods are explained in detail using three phylogenetically diverse marine invertebrates: Actinia equina (Anthozoa, Cnidaria), Harmothoe sp. (Polychaeta, Annelida), and Xenoturbella japonica (Xenoturbellida, Xenacoelomorpha). Suggestions on performing microCT imaging on various animals are also provided.

Microfocus X-ray CT microCT Imaging of Actinia equina Cnidaria, Harmothoe sp. Annelida, and Xenoturbella japonica Xenacoelomorpha

Here, protocols for performing microfocus X-ray computed tomography (microCT) imaging of three marine invertebrate animals are explained in detail. This study describes steps such as sample fixation, staining, mounting, scanning, image reconstruction, and data analyses. Suggestions on how the protocol can be adjusted for different samples are also provided.

Traditionally, biologists have had to rely on destructive methods such as sectioning in order to investigate the internal structures of opaque organisms. ...

Environment

Hybrid &#181;CT-FMT imaging and image analysis

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to assess the biodistribution of novel probes and to assess disease progression using established molecular probes or reporter genes. The combination with an anatomical modality, e.g., micro computed tomography (&#181;CT), is beneficial for image analysis and for fluorescence reconstruction. We describe a protocol for multimodal &#181;CT-FMT imaging including the image processing steps necessary to extract quantitative measurements. After preparing the mice and performing the imaging, the multimodal data sets are registered. Subsequently, an improved fluorescence reconstruction is performed, which takes into account the shape of the mouse. For quantitative analysis, organ segmentations are generated based on the anatomical data using our interactive segmentation tool. Finally, the biodistribution curves are generated using a batch-processing feature. We show the applicability of the method by assessing the biodistribution of a well-known probe that binds to bones and joints.

Hybrid  &#181;CT-FMT imaging and image analysis

We describe a protocol for hybrid imaging, combining fluorescence-mediated tomography (FMT) with micro computed tomography (&#181;CT). After fusion and reconstruction, we perform interactive organ segmentation to extract quantitative measurements of the fluorescence distribution.

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to ...

Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This enables quantifications of ultrastructural aspects such as volume fractions, surface area to volume ratios, morphometry, and physical contact sites of different subcellular structures. In the 1970s, a protocol for enhanced staining of glycogen in cells was developed and paved the way for a string of studies on the subcellular localization of glycogen and glycogen particle size using transmission electron microscopy. While most analyses interpret glycogen as if it is homogeneously distributed within the muscle fibers, providing only a single value (e.g., an average concentration), transmission electron microscopy has revealed that glycogen is stored as discrete glycogen particles located in distinct subcellular compartments. Here, the step-by-step protocol from tissue collection to the quantitative determination of the volume fraction and particle diameter of glycogen in the distinct subcellular compartments of individual skeletal muscle fibers is described. Considerations on how to 1) collect and stain tissue specimens, 2) perform image analyses and data handling, 3) evaluate the precision of estimates, 4) discriminate between muscle fiber types, and 5) methodological pitfalls and limitations are included.

A modified post-fixation procedure increases the contrast of glycogen particles in tissue. This paper provides a step-by-step protocol describing how to handle the tissue, conduct the imaging, and use stereological methods to obtain unbiased and quantitative data on fiber type-specific subcellular glycogen distribution in skeletal muscle.

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This ...

Biochemistry

A Guide to Examining Intramuscular Fat Formation and its Cellular Origin in Skeletal Muscle

Fibro-adipogenic progenitors (FAPs) are mesenchymal stromal cells that play a crucial role during skeletal muscle homeostasis and regeneration. FAPs build and maintain the extracellular matrix that acts as a molecular myofiber scaffold. In addition, FAPs are indispensable for myofiber regeneration as they secrete a multitude of beneficial factors sensed by the muscle stem cells (MuSCs). In diseased states, however, FAPs are the cellular origin of intramuscular fat and fibrotic scar tissue. This fatty fibrosis is a hallmark of sarcopenia and neuromuscular diseases, such as Duchenne Muscular Dystrophy. One significant barrier in determining why and how FAPs differentiate into intramuscular fat is effective preservation and subsequent visualization of adipocytes, especially in frozen tissue sections. Conventional methods of skeletal muscle tissue processing, such as snap-freezing, do not properly preserve the morphology of individual adipocytes, thereby preventing accurate visualization and quantification. To overcome this hurdle, a rigorous protocol was developed that preserves adipocyte morphology in skeletal muscle sections allowing visualization, imaging, and quantification of intramuscular fat. The protocol also outlines how to process a portion of muscle tissue for RT-qPCR, enabling users to confirm observed changes in fat formation by viewing differences in the expression of adipogenic genes. Additionally, it can be adapted to visualize adipocytes by whole-mount immunofluorescence of muscle samples. Finally, this protocol outlines how to perform genetic lineage tracing of Pdgfr&#945;-expressing FAPs to study the adipogenic conversion of FAPs. This protocol consistently yields high-resolution and morphologically accurate immunofluorescent images of adipocytes, along with confirmation by RT-qPCR, allowing for robust, rigorous, and reproducible visualization and quantification of intramuscular fat. Together, the analysis pipeline described here is the first step to improving our understanding of how FAPs differentiate into intramuscular fat, and provides a framework to validate novel interventions to prevent fat formation.

The replacement of healthy muscle tissue with intramuscular fat is a prominent feature of human diseases and conditions. This protocol outlines how to visualize, image, and quantify intramuscular fat, allowing the rigorous study of the mechanisms underlying intramuscular fat formation.

Fibro-adipogenic progenitors (FAPs) are mesenchymal stromal cells that play a crucial role during skeletal muscle homeostasis and regeneration. FAPs build ...

Developmental Biology

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

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 ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

Medicine

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and geometric&#160;deformation. Our understanding of the internal relationships between these events has been limited because we can neither &#39;see&#39; inside the muscle nor precisely track the spatio-temporal nature of excitation-contraction dynamics. To resolve these problems, we have constructed a device that combines a suite of imaging modalities. Specifically, it integrates a brightfield microscope to measure local changes of sarcomere length and tissue strain, a fluorescence microscope to visualize the Ca2+ transient, and an optical coherence tomograph to capture the tissue&#39;s geometric&#160;changes throughout the time-course of a cardiac cycle. We present here the imaging infrastructure and associated data collection framework. Data are collected from isolated rod-like tissue structures known as trabeculae carneae. In our instrument, a pair of position-controlled platinum hooks hold each end of an ex vivo muscle sample while it is continuously superfused with nutrient-rich saline solution. The hooks are under independent control, permitting real-time control of muscle length and force. Lengthwise translation enables the piecewise scanning of the sample, overcoming limitations associated with the relative size of the microscope imaging window (540 &#181;m by 540 &#181;m) and the length of a typical trabecula (&#62;2000 &#181;m). Platinum electrodes at either end of the muscle chamber stimulate the trabecula at a user-defined rate. We exploit the stimulation signal as a trigger for synchronizing the data from each imaging window to reconstruct the entire sample twitching under steady-state conditions. Applying image-processing techniques to these brightfield imaging data provides tissue displacement and sarcomere length maps. Such a collection of data, when incorporated into an experiment-modeling pipeline, will provide a deeper understanding of muscle contractile homogeneity and heterogeneity in physiology and pathophysiology.

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

This protocol presents a collection of sarcomere, calcium, and macroscopic geometric&#160;data from an actively contracting cardiac trabecula ex vivo. These simultaneous measurements are made possible by the integration of three imaging modalities.

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and ...

Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional properties of diseased tissue is critical to determine their interactions with arrhythmic behavior. Microscale tissue remodeling, below the clinical resolution, is emerging as an important source of lethal arrhythmia, with high prevalence in young adults. Challenges remain in obtaining high imaging contrast at sufficient microscale resolution for preclinical models, such as large mammalian whole hearts. Moreover, tissue composition-selective contrast enhancement for three-dimensional high-resolution imaging is still lacking. Non-destructive imaging using micro-computed tomography shows promise for high-resolution imaging. The objective was to alleviate sufferance from X-ray over attenuation in large biological samples. Hearts were extracted from healthy pigs (N = 2), and sheep (N = 2) with either induced chronic myocardial infarction and fibrotic scar formation or induced chronic atrial fibrillation. Excised hearts were perfused with: a saline solution supplemented with a calcium ion quenching agent and a vasodilator, ethanol in serial dehydration, and hexamethyldisilizane under vacuum. The latter reinforced the heart structure during air-drying for 1 week. Collagen-dominant tissue was selectively bound by an X-ray contrast-enhancing agent, phosphomolybdic acid. Tissue conformation was stable in air, permitting long-duration microcomputed tomography acquisitions to obtain high-resolution (isotropic 20.7 &#181;m) images. Optimal contrast agent loading by diffusion showed selective contrast enhancement of the epithelial layer and sub-endocardial Purkinje fibers in healthy pig ventricles. Atrial fibrillation (AF) hearts showed enhanced contrast accumulation in the posterior walls and appendages of the atria, attributed to greater collagen content. Myocardial infarction hearts showed increased contrast selectively in regions of cardiac fibrosis, which enabled the identification of interweaving surviving myocardial muscle fibers. Contrast-enhanced air-dried tissue preparations enabled microscale imaging of the intact large mammalian heart and selective contrast enhancement of underlying disease constituents.

Here, we present a protocol to obtain high resolution micro computed tomography images of healthy and pathological large mammalian whole hearts with collagen-selective contrast enhancement.

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional ...

Sample Preparation for Computed Tomography-based Three-dimensional Visualization of Murine Hind-limb Vessels

Blood vessels are complex networks with tree-like structures, and vascular networks are essential for maintaining both circulation and maintaining organ function. Clarifying the mechanism of blood vessel formation is therefore extremely useful for elucidating developmental processes and pathological mechanisms. Murine hind-limb vessels are often used as a model for physiological and pathological angiogenesis. Evaluation is mainly performed via a two-dimensional method using tissue sections. However, methods for evaluating three-dimensional (3D) vascular morphology are particularly limited. This paper introduces a method for visualizing murine hind-limbs using computed tomography (CT). Radiation-opaque resin is injected through the descending aorta, and whole vessels are filled with dye. By adjusting the time of dye injection, arterial-specific filling is also possible, and samples can be obtained with any micro-X-ray CT device. This contrast method provides a basic technique for the 3D evaluation of murine blood vessels in the lower extremities. Furthermore, this method can be used to visualize all blood vessels below the diaphragm and evaluate blood vessels in the abdominal organs.

Here, we describe a visualization and quantification method for murine hind-limb vessels using micro-X-ray computed tomography.

Blood vessels are complex networks with tree-like structures, and vascular networks are essential for maintaining both circulation and maintaining organ ...

Use of Micro X-ray Computed Tomography with Phosphotungstic Acid Preparation to Visualize Human Fibromuscular Tissue

Samenvatting

Meer video's verkennen