We would like to thank Toshihiko Shiroishi for his assistance and for providing the research environment during this study. We are grateful to Kensuke Yanagi and Takato Izumi for advice on A. equina, and Masaatsu Tanaka for advice on the Harmothoe sp. specimen. We would like to thank the staff at Shimoda Marine Research Center, University of Tsukuba, and Misaki Marine Biological Station, The University of Tokyo for their help in sample collections. We would like to thank Editage (www.editage.jp) for English language editing. This work was supported by the JSPS Grant-in-Aid for Young Scientists (A) (JP26711022) to HN, and JAMBIO, Japanese Association for Marine Biology.

1. Fixation
<ol>
	<li>For Actinia equina, relax the animals in 10% MgCl2 seawater for about 15 min at room temperature. Transfer to 70% ethanol and store at room temperature.</li>
	<li>For Harmothoe sp., anesthetize the animals by placing them in ice-cold seawater for about 15 min. Transfer to 10% (v/v) formalin solution with seawater and store at room temperature.</li>
	<li>For Xenoturbella japonica, relax the animal using 7% MgCl2 in freshwater. Fix in 4% paraformaldehyde (PFA) in filtered seawater overnight. Place in 70% ethanol and store at 4 &#176;C. 
	CAUTION: PFA is hazardous and must be handled with care.</li>
</ol>
2. Staining
<ol>
	<li>Transfer the samples to 50% ethanol and store at room temperature for 15 h. Replace the 50% ethanol with 25% ethanol and store at room temperature for 2 h. 
	NOTE: This is not necessary for the Harmothoe sp. sample in 10% (v/v) formalin solution with seawater.</li>
	<li>Replace the solution with distilled water (DW) and store the samples in DW at room temperature for 2 h. Repeat this step three times.</li>
	<li>Prepare 25% Lugol solution by diluting the stock solution (below) to 25% with DW. Stock solution (100% Lugol solution) contains 10 g of KI and 5 g of I2, adjusted to 100 mL with DW. 
	NOTE: Lugol solution is light-sensitive, so store and handle the solution protected from light. Follow the regulations of each country and institution for iodine handling and waste disposal.</li>
	<li>Decant the DW from the samples and pour in the 25% Lugol solution. Stain for 24 h at room temperature.</li>
</ol>
3. Stage Mounting
<ol>
	<li>Prepare 0.5% agarose by dissolving 500 mg agarose in 100 mL of DW in a 250 mL conical flask in a microwave (800 W, about 1-3 min). Cool to about 30-40 &#176;C by keeping at room temperature. 
	CAUTION: Do not overheat or completely seal the flask when heating to prevent the agarose from boiling over.</li>
	<li>Mount large&#160;samples such as A. equina using a 50 mL tube. 
	NOTE: Use 50 mL tubes for mounting large samples that do not fit in a 1,000 &#956;L micropipette &#39;blue&#39; tip.
	<ol>
		<li>Place the stained sample in a 60-mm dish with DW to wash off excessive staining solution from the surface.</li>
		<li>Gently pour 5 mL of 0.5% agarose into a 50 mL tube and harden the agarose on ice. Be careful not to make bubbles in the agarose.</li>
		<li>Gently add 20 mL of 0.5% agarose to the 50 mL tube and place on ice until the agarose begins to harden. Place the specimen within the 0.5% agarose using forceps. Be careful not to make bubbles in the agarose.</li>
		<li>Adjust the position and orientation of the sample with forceps and harden the agarose on ice.</li>
		<li>Place clay on the microCT mounting stage and set the 50 mL tube on the clay (Figure 2A).</li>
	</ol>
	</li>
	<li>Mount small&#160;samples such as Harmothoe sp. and X. japonica using a 1,000 &#181;L micropipette &#39;blue&#39;&#160;tip. 
	NOTE: For smaller samples, 200 &#956;L micropipette &#8216;yellow&#8217; tip can be used. In this case, use 30 &#956;L of agarose for the plug and add 200 &#956;L of agarose or distilled water.
	<ol>
		<li>Draw up 100 &#181;L of 0.5% agarose into a 1,000 &#181;L micropipette &#39;blue&#39;&#160;tip and harden the agarose by holding the tip over&#160;ice, making a plug in the tip (Figure 2B-a).</li>
		<li>Decant the stained sample into a 60-mm dish without using forceps.</li>
		<li>Gently transfer the sample using ring tweezers into another 60-mm dish with DW to wash off excessive staining solution from the surface.</li>
		<li>Add 1,000 &#181;L of either&#160;0.5% agarose or&#160;DW into the plugged tip (step 3.3.1) using a micropipette. 
		NOTE: Use of 0.5% agarose is recommended, but use DW for fragile or precious samples in which agarose should be avoided.</li>
		<li>Gently transfer the sample from the 60-mm dish into the agarose or&#160;DW in the plugged tip using ring tweezers.</li>
		<li>Gently adjust the position and orientation of the sample with a petiolate needle or precision tweezers. Be careful not to make bubbles in the mounting medium. Make sure the sample is stable between the walls of the tip when DW is used as mounting medium (Figure 2D-b). Place the tip on ice to harden the agarose if it is used as mounting medium.</li>
		<li>Cut the tip off a new 1,000 &#181;L micropipette &#39;blue&#39;&#160;tip (Figure 2B-b,c) and insert the tip of the plugged tip into the new tip.</li>
		<li>Place clay on the microCT mounting stage and set the tips with the sample inside on the clay (Figure 2C,D). 
		NOTE: The staining solution will start to wash off the sample once it is placed in DW, so proceed to the next scanning step promptly.</li>
	</ol>
	</li>
</ol>
4. MicroCT scanning
<ol>
	<li>Turn on the X-ray beam at 80 kV, 100 &#181;A.</li>
	<li>While observing the X-ray transmission image at the center of the screen (Figure 3A), move the stage so that the whole sample can be seen by clicking on the X and Z axis buttons (Figure 3A), and manually adjusting the Y axis knob on the mounting stage (Figure 3B). Set the contrast of the image so that the internal structures can be observed by adjusting the contrast conditions (Figure 3A: Image contrast).</li>
	<li>Adjust the orientation of the sample by changing the angle of the tube/tip in the clay (Figure 2A). Rotate the stage 90&#176; by setting the rotation axis (Figure 3A) to 90 and clicking on the relative movement button (Figure 3A). Perform the same maneuver four times to complete a full rotation. 
	NOTE: Manually turn off the X-ray beam each time the sample door is opened, unless the system turns it off automatically.</li>
	<li>Move the stage so that the sample is at the center of the view by clicking on the Z axis button (Figure 3A) and by manually adjusting the Y axis knob on the mounting stage (Figure 3B). Turn the stage by 90&#176; and do the same. Turn the stage 360&#176; and check that the sample is at the center of the view from all directions.</li>
	<li>Move the stage along the x-axis toward the X-ray beam source by clicking on the X axis button (Figure 3A) to enlarge the sample and adjust as needed so that it just fits in the view (Figure 3C).</li>
	<li>Turn the stage 360&#176; and adjust as needed so&#160;that the sample fits within view from all directions.</li>
	<li>Adjust the scanning conditions as shown in Table 1.</li>
	<li>Start scanning; it takes about 10 min.</li>
</ol>
5. Image reconstruction
<ol>
	<li>Start up the microCT system&#39;s accessory software (see the Table of Materials) and open the scanned data.</li>
	<li>Adjust differences in the rotation axis of the sample during scanning by clicking on the automatic shift value calculation button (Figure 4A: green box).</li>
	<li>Adjust the orientation of the image by rotating the orange arrows (Figure 4B). If the orientation was changed, repeat step 5.2.</li>
	<li>Click on the area tab (Figure 4C: magenta box) and trim areas where samples are not present (Figure 4C: yellow box).</li>
	<li>Click on the reconfiguration tab (Figure 4D: magenta box) and set the filters as follows to remove noise. Ring artifact reduce filter: Median filter -3; Noise elimination filter: Average filter-1.</li>
	<li>Perform reconfiguration by clicking on the reconfiguration button (Figure 4D: green box).</li>
	<li>Adjust image brightness and contrast by setting the black and white values as black value 0, white value 250 (Figure 4D: blue box).</li>
	<li>Save the reconstructed TIFF image dataset as an 8-bit TIFF by clicking on the save button. Rename TIFF files as following: Date_sample_resolution (&#181;m)_number.tiff. 
	NOTE: The original microCT datasets from this study are available in the Figshare repository, doi:10.6084/m9.figshare.767083736.</li>
</ol>
6. Data analyses
<ol>
	<li>Start up the data analysis software (see the Table of Materials) and enable importing of TIFF files by clicking on the Database icon (Figure 5A: magenta box) and turning off the box shown in Figure 5B.</li>
	<li>Click on the import icon (Figure 5C: magenta box), select the dataset saved in section 5, and click open.</li>
	<li>Click on the copy links button (Figure 5D) to import the data.</li>
	<li>Display the 2D cross-section by clicking on the 2D viewer icon (Figure 5C: blue box).</li>
	<li>Calibrate the dataset by clicking on the 3D viewer tab (Figure 5E: magenta box) and entering the resolution value at scanning (which was 0.018 in this study [Figure 5F]).</li>
	<li>Click on the brightness/contrast icon (Figure 5E green box). Adjust the brightness and contrast by moving the cursor inside the displayed 2D image and changing the window level and window width (Figure 5G).</li>
	<li>Check other cross-section images by moving the scrollbar (Figure 5G: box).</li>
	<li>Change the orientation of the cross-section by clicking on the orientation icon (Figure 5E: blue box) and check images at all orientations (Figure 5H).</li>
	<li>Click on the displayed image, and choose the export tab to store cross-section images.</li>
</ol>

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D., Giribet, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+micro-computed+tomography+as+a+minimally+invasive+tool+for+anatomical+study+of+bivalves+(Mollusca:+Bivalvia).">The use of micro-computed tomography as a minimally invasive tool for anatomical study of bivalves (Mollusca: Bivalvia).</a> Zoological Journal of the Linnean Society. , (2018).</li><li>Sasaki, T., Endo, K., Kogure, T., Nagasawa, H., 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=3D+visualization+of+calcified+and+non-calcified+molluscan+tissues+using+computed+tomography.">3D visualization of calcified and non-calcified molluscan tissues using computed tomography.</a> Biomineralization. , 83-93 (2018).</li><li>Maeno, A., Tsuda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-computed+Tomography+to+Visualize+Vascular+Networks+in+Maize+Stems.">Micro-computed Tomography to Visualize Vascular Networks in Maize Stems.</a> Bio-protocol. 8 (1), 2682 (2018).</li><li>Nakano, H., 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=Correction+to:+A+new+species+of+Xenoturbella+from+the+western+Pacific+Ocean+and+the+evolution+of+Xenoturbella.">Correction to: A new species of Xenoturbella from the western Pacific Ocean and the evolution of Xenoturbella.</a> BMC Evolutionary Biology. 18, 83 (2018).</li><li>Maeno, A., Kohtsuka, H., Takatani, K., Nakano, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroCT+files+from+'Microfocus+X-ray+computed+tomography+(microCT)+imaging+of+Actinia+equina+(Cnidaria),+Harmothoe+sp.+(Annelida),+and+Xenoturbella+japonica+(Xenacoelomorpha)'.">MicroCT files from 'Microfocus X-ray computed tomography (microCT) imaging of Actinia equina (Cnidaria), Harmothoe sp. (Annelida), and Xenoturbella japonica (Xenacoelomorpha)'.</a> figshare. , (2019).</li><li>Vickerton, P., Jarvis, J., Jeffery, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration-dependent+specimen+shrinkage+in+iodine-enhanced+microCT.">Concentration-dependent specimen shrinkage in iodine-enhanced microCT.</a> Journal of Anatomy. 223 (2), 185-193 (2013).</li><li>Buytaert, J., Goyens, J., De Greef, D., Aerts, P., Dirckx, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volume+shrinkage+of+bone,+brain+and+muscle+tissue+in+sample+preparation+for+micro-CT+and+light+sheet+fluorescence+microscopy+(LSFM).">Volume shrinkage of bone, brain and muscle tissue in sample preparation for micro-CT and light sheet fluorescence microscopy (LSFM).</a> Microscopy and Microanalysis. 20 (4), 1208-1217 (2014).</li><li>Sasov, A., Liu, X., Salmon, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compensation+of+mechanical+inaccuracies+in+micro-CT+and+nano-CT.">Compensation of mechanical inaccuracies in micro-CT and nano-CT.</a> Proceedings of SPIE. 7078, 70781 (2008).</li></ol>

The authors have nothing to disclose.

Fixatives using formalin, such as the 10% (v/v) formalin solution in seawater used in this study, are known to preserve the morphology of diverse marine invertebrates and are often used for microCT imaging18,24,25,26,28,30,33. However, restrictions on the use of this chemical have become str...

Biological researchers generally have had to make thin sections and perform observations by light or electron microscopy in order to investigate the internal structures of opaque organisms. However, these methods are destructive and problematic when applied to rare or valuable specimens. Furthermore, several steps in the method, such as embedding and sectioning, are time consuming, and it can take several days to observe a sample, depending on the protocol. Moreover, when handling numerous sections, there is always a possibility of damaging or losing some sections. Tissue-clearing techniques are available for some specimens1,2,3,4,5 but are not yet applicable for many animal species.
To overcome these problems, some biologists have started using microfocus X-ray computed tomography (microCT) imaging6,7,8,9,10,11,12,13,14,15. In X-ray CT, the specimen is irradiated with X-rays from various angles that are generated from an X-ray source moving around the sample, and the transmitted X-rays are monitored by a detector that also moves around the sample. The X-ray transmission data obtained are analyzed to reconstruct cross-sectional images of the specimen. This method enables the observation of internal structures without destruction of the sample. Because of its safety and ease, it is commonly used in medical and dental applications, and CT systems can be found in hospitals and dental centers worldwide. Moreover, industrial X-ray CT is frequently used for observing non-medical samples for inspection and metrology in the industrial field. In contrast to medical CT, in which the X-ray source and the detectors are mobile, the two parts are fixed in industrial CT, with the sample rotating during scanning. Industrial CT generally produces higher resolution images than medical CT and is referred to as microCT (micrometer-level resolution) or nanoCT (nanometer-level resolution). Recently, research using microCT has rapidly increased in various fields of biology14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34.
Biological studies using CT initially targeted internal structures that mainly consist of hard tissue, such as bone. Advances in staining techniques using various chemical agents enabled the visualization of soft tissues in various organisms6,7,8,9,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34. Of these reagents, iodine-based contrast agents are relatively safe, inexpensive, and can be used for the visualization of soft tissues in various organisms7,14. Concerning marine invertebrates, microCT has been widely used on such animals as molluscs6,25,32,33, annelids18,19,20,28, and arthoropods21,23,29,31. However, there have been few reports on other animal phyla, such as bryozoans6, xenacoelomorphs26, and cnidarians24,30. In general, there have been fewer studies using microCT on marine invertebrates than those on vertebrates. One major reason for this limited use on marine invertebrates is the vast diversity observed in these animals. Because of their diverse sizes, morphologies, and physiologies, each species reacts differently to different experimental procedures. Therefore, it is crucial during sample preparation to choose the most appropriate fixation and staining reagent, and to set conditions at each step, adjusted for each species. Similarly, it is also necessary to set the scanning configurations, such as the mounting method, voltage, current, mechanical magnifying rate, and the space resolution power, appropriately for each sample. To overcome this problem, a simple and comprehensible manual that covers all of the necessary steps, explains how each step can be adjusted depending on the specimen, and shows detailed examples from multiple samples is essential.
In the present study, we describe the microCT imaging protocol step-by-step, from sample fixation to data analysis, using three marine invertebrate species. Specimens of the sea anemone Actinia equina (Anthozoa, Cnidaria) were collected near the Misaki Marine Biological Station, University of Tokyo. They had a spherical, soft body that was about 2 cm in diameter (Figure 1A-C). Harmothoe sp. (Polychaeta, Annelida) samples were also collected near Misaki Marine Biological Station. They were slender worms that were about 1.5 cm in length, with tough chaetae present along the whole body (Figure&#160;1D). A Xenoturbella japonica35 (Xenoturbellida, Xenacoelomorpha) specimen was collected near Shimoda Marine Research Center, University of Tsukuba, during the 13th JAMBIO Coastal Organism Joint Survey. It was a soft-bodied worm that was about 0.8 cm in length (Figure 1E). Adjustments made for the conditions and configurations of each sample are explained in detail. Our study provides several suggestions on how to perform microCT imaging on marine invertebrates, and we hope that it will inspire biologists to utilize this protocol for their research.

<table><tbody><tr><td>250-ml Erlenmeyer flask</td><td>Corning</td><td>CLS430183</td><td></td></tr><tr><td>5-ml Sampling tube ST-500</td><td>BIO-BIK</td><td>103010</td><td></td></tr><tr><td>50-ml Polypropylene tube</td><td>Greiner Bio One International</td><td>227261</td><td></td></tr><tr><td>60-mm Non-treated Dish</td><td>IWAKI</td><td>1010-060</td><td></td></tr><tr><td>Agarose</td><td>Promega</td><td>V3125</td><td></td></tr><tr><td>Ecological grade tip (blue) 1000 &amp;micro;l</td><td>BMBio</td><td>BIO1000RF</td><td></td></tr><tr><td>Ethanol</td><td>Wako Pure Chemical Industries</td><td>057-00451</td><td></td></tr><tr><td>Formalin</td><td>Wako Pure Chemical Industries</td><td>061-00416</td><td></td></tr><tr><td>Iodine</td><td>Wako Pure Chemical Industries</td><td>094-05421</td><td></td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Wako Pure Chemical Industries</td><td>135-00165</td><td></td></tr><tr><td>OsiriX DICOM Viewer</td><td>Pixmeo SARL</td><td>OsiriX MD v10.0</td><td>https://www.osirix-viewer.com</td></tr><tr><td>Paraformaldehyde</td><td>Wako Pure Chemical Industries</td><td>163-25983</td><td></td></tr><tr><td>Petiolate needle</td><td>AS ONE</td><td>2-013-01</td><td></td></tr><tr><td>Pipetman P200 Micropipette</td><td>GILSON</td><td>F123601</td><td></td></tr><tr><td>Pipetman P1000 Micropipette</td><td>GILSON</td><td>F123602</td><td></td></tr><tr><td>Potassium iodide</td><td>Wako Pure Chemical Industries</td><td>166-03971</td><td></td></tr><tr><td>Precision tweezers 5</td><td>DUMONT</td><td>0302-5-PS</td><td></td></tr><tr><td>QuickRack MultI fit tip (yellow) 200 ul</td><td>Sorenson</td><td>10660</td><td></td></tr><tr><td>Razor blades</td><td>Feather</td><td>FA-10</td><td></td></tr><tr><td>Ring tweezers</td><td>NAPOX</td><td>A-26</td><td></td></tr><tr><td>Stereoscopic microscope</td><td>Leica</td><td>MZ95</td><td></td></tr><tr><td>X-ray Micro-CT imaging system</td><td>Comscantechno</td><td>ScanXmate-E090S105</td><td></td></tr></tbody></table>

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.

We performed microCT imaging on A. equina (Anthozoa, Cnidaria), Harmothoe sp. (Polychaeta, Annelida), and X. japonica (Xenoturbellida, Xenacoelomorpha) after staining the samples with 25% Lugol solution. The staining successfully enhanced the contrast of the internal structures in all specimens, enabling observations of internal soft tissues (Figure 6). Together with past reports6,7,<sup class="x...

Watch this Scientific Journal Video about Microfocus X-ray CT microCT Imaging of Actinia equina Cnidaria, Harmothoe sp. Annelida, and Xenoturbella japonica Xenacoelomorpha at JoVE.com

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.

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. ...

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Research

JoVE Journal

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9.0K Views.  National Institute of Genetics. 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.

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

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

Contrast Enhanced Vessel Imaging using MicroCT

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between different soft tissues and body fluids is inherently poor in micro-CT images 1. Under these circumstances, visualization of blood vessels becomes a nearly impossible task. To overcome this and to improve the visualization of blood vessels exogenous contrast agents can be used. Herein, we present a methodology for visualizing the vascular network in a rodent model. By using a long-acting aqueous colloidal polydisperse iodinated blood-pool contrast agent, eXIA 160XL, we optimized image acquisition parameters and volume-rendering techniques for finding blood vessels in live animals. Our findings suggest that, to achieve a superior contrast between bone and soft tissue from vessel, multiple-frames (at least 5-8/ frames per view), and 360-720 views (for a full 360&deg; rotation) acquisitions were mandatory. We have also demonstrated the use of a two-dimensional transfer function (where voxel color and opacity was assigned in proportion to CT value and gradient magnitude), in visualizing the anatomy and highlighting the structure of interest, the blood vessel network. This promising work lays a foundation for the qualitative and quantitative assessment of anti-angiogenesis preclinical studies using transgenic or xenograft tumor-bearing mice.

Contrast enhanced small animal vessel imaging by microCT is a rapid, cost-effective and high-throughput technique for serial in situ examination for tumor development, for analyzing the network of blood vessels that nourish them, and for following the response of tumors to preclinical therapeutic intervention(s).

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between ...

Medicine

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

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

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

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

Biology

Characterization Of Multi-layered Fish Scales (Atractosteus spatula) Using Nanoindentation, X-ray CT, FTIR, and SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle shells provides unique design principles with potentials for guiding the design of protective materials and systems in the future. Understanding the structure-property relationships for these material systems at the microscale and nanoscale where failure initiates is essential. Currently, experimental techniques such as nanoindentation, X-ray CT, and SEM provide researchers with a way to correlate the mechanical behavior with hierarchical microstructures of these material systems1-6. However, a well-defined standard procedure for specimen preparation of mineralized biomaterials is not currently available. In this study, the methods for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of A. spatula using nanoindentation, FTIR, SEM, with energy-dispersive X-ray (EDX) microanalysis, and X-ray CT are presented.

Characterization Of Multi-layered Fish Scales Atractosteus spatula Using Nanoindentation, X-ray CT, FTIR, and SEM

This paper presents the methods used for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of Atractosteus spatula (A. spatula) using nanoindentation, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and X-ray computed tomography (X-ray CT). The experimental results have been used to investigate the design principles of protective biological materials.

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle ...

Leveraging Micro-CT Scanning to Analyze Parasitic Plant-Host Interactions

Micro-CT scanning has become an established tool in investigating plant structure and function. Its non-destructive nature, combined with the possibility of three-dimensional visualization and virtual sectioning, has allowed novel and increasingly detailed analysis of complex plant organs. Interactions among plants, including between parasitic plants and their hosts, can also be explored. However, sample preparation before scanning becomes crucial due to the interaction between these plants, which often differ in tissue organization and composition. Furthermore, the broad diversity of parasitic flowering plants, ranging from highly reduced vegetative bodies to trees, herbs, and shrubs, must be considered during the sampling, treatment, and preparation of parasite-host material. Here two different approaches are described for introducing contrast solutions into the parasite and/or host plants, focusing on analyzing the haustorium. This organ promotes connection and communication between the two plants. Following a simple approach, details of haustorium tissue organization can be explored three-dimensionally, as shown here for&#160;euphytoid, vine, and mistletoe parasitic species. Selecting specific contrasting agents and application approaches also allow detailed observation of endoparasite spread within the host body and detection of direct vessel-to-vessel connection between parasite and host, as shown here for an obligate root parasite. Thus, the protocol discussed here can be applied to the broad diversity of parasitic flowering plants to advance the understanding of their development, structure, and functioning.

Micro-CT is a non-destructive tool that can analyze plant structures in three dimensions. The present protocol describes the sample preparation to leverage micro-CT to analyze parasitic plant structure and function. Different species are used to highlight the advantages of this method when coupled with specific preparations.

Micro-CT scanning has become an established tool in investigating plant structure and function. Its non-destructive nature, combined with the possibility ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse models of lung cancer are being used to study the mechanism of cancer development and to experiment with various therapeutic strategies. However, the absence of a real-time technique to identify the development of tumor nodules in mice lungs and to monitor the changes in their size in response to various experimental and therapeutic interventions hampers the ability to obtain an accurate description of the course of the disease and its timely response to treatments. In this study, a method using a micro-computed tomography (CT) scanner for the detection of the development of lung tumors in a mouse model of lung adenocarcinoma is described. Next, we show that monthly follow-up with micro-CT can identify dynamic changes in the lung tumor, such as the appearance of additional nodules, increase in the size of previously detected nodules, and decrease in the size or complete resolution of nodules in response to treatment. Finally, the accuracy of this real-time assessment method was confirmed with end-point histological quantification. This technique paves the way for planning and conducting more complex experiments on lung cancer animal models, and it enables us to better understand the mechanisms of carcinogenesis and the effects of different treatment modalities while saving time and resources.

We describe a method for the detection of tumor nodule development in the lungs of an adenocarcinoma mouse model using micro-computed tomography and its use for monitoring changes in nodule size over time and in response to treatment. The accuracy of the assessment was confirmed with end-point histological quantification.

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse ...

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.

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 ...

In Vitro Preparation of Actively Maturing Bovine Articular Cartilage Explants for X-Ray Phase Contrast Imaging

Understanding the mechanisms that underpin post-natal maturation of articular cartilage is of crucial importance for designing the next generation of tissue engineering strategies and potentially repairing diseased or damaged cartilage. In general, postnatal maturation of the articular cartilage, which is a wholesale change in collagen structure and function of the tissue to accommodate growth of the organism, occurs over a timescale ranging from months to years. Conversely dissolution of the structural organization of the cartilage that also occurs over long timescales is the hallmark of tissue degeneration. Our ability to study these biological processes in detail have been enhanced by the findings that growth factors can induce precocious in vitro maturation of immature articular cartilage. The developmental and disease related changes that occur in the joint involve bone and cartilage and an ability to co-image these tissues would significantly increase our understanding of their intertwined roles.
The simultaneous visualization of soft tissue, cartilage and bone changes is nowadays a challenge to overcome for conventional preclinical imaging modalities used for the joint disease follow-up. Three-dimensional X-ray Phase-Contrast Imaging methods (PCI) have been under perpetual developments for 20 years due to high performance for imaging low density objects and their ability to provide additional information compared to conventional X-ray imaging.
In this protocol we detail the procedure used in our experiments from biopsy of the cartilage, generation of in vitro matured cartilage to data analysis of image collected using X-ray phase contrast imaging.

This protocol describes the preparation of in vitro bovine articular cartilage for imaging in high resolution with X-rays. These explants actively undergo postnatal maturation. We describe here the necessary steps from the biopsy to data analysis of 3D X-ray phase contrast imaging, passing through explant culture, tissue fixation and synchrotron preparation.

Understanding the mechanisms that underpin post-natal maturation of articular cartilage is of crucial importance for designing the next generation of ...

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 ...

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