Name: microfocus x-ray ct (microct) imaging of actinia equina (cnidaria), harmothoe sp. (annelida), and xenoturbella japonica (xenacoelomorpha)
Uploaded: 2019-08-06T14:30:00
Description: 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

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% paraformaldeh...

<ol>
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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.

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			<td height="21" style="height:21px;width:264px;">250-ml Erlenmeyer flask</td>
			<td style="width:223px;">Corning</td>
			<td style="width:163px;">CLS430183</td>
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		<tr height="21">
			<td height="21" style="height:21px;">5-ml Sampling tube ST-500</td>
			<td>BIO-BIK</td>
			<td>103010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50-ml Polypropylene tube</td>
			<td>Greiner Bio One International</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60-mm Non-treated Dish</td>
			<td>IWAKI</td>
			<td>1010-060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td>Promega</td>
			<td>V3125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ecological grade tip (blue) 1000 &#181;l</td>
			<td>BMBio</td>
			<td>BIO1000RF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>Wako Pure Chemical Industries</td>
			<td>057-00451</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formalin</td>
			<td>Wako Pure Chemical Industries</td>
			<td>061-00416</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iodine</td>
			<td>Wako Pure Chemical Industries</td>
			<td>094-05421</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium chloride hexahydrate</td>
			<td>Wako Pure Chemical Industries</td>
			<td>135-00165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OsiriX DICOM Viewer</td>
			<td>Pixmeo SARL</td>
			<td>OsiriX MD v10.0</td>
			<td>https://www.osirix-viewer.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Wako Pure Chemical Industries</td>
			<td>163-25983</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petiolate needle</td>
			<td>AS ONE</td>
			<td>2-013-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipetman P200 Micropipette</td>
			<td>GILSON</td>
			<td>F123601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipetman P1000 Micropipette</td>
			<td>GILSON</td>
			<td>F123602</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium iodide</td>
			<td>Wako Pure Chemical Industries</td>
			<td>166-03971</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Precision tweezers 5</td>
			<td>DUMONT</td>
			<td>0302-5-PS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QuickRack MultI fit tip (yellow) 200 ul</td>
			<td>Sorenson</td>
			<td>10660</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Razor blades</td>
			<td>Feather</td>
			<td>FA-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ring tweezers</td>
			<td>NAPOX</td>
			<td>A-26</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereoscopic microscope</td>
			<td>Leica</td>
			<td>MZ95</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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. ...

Micro CT has not been widely applied to marine invertebrates due to their diversity in morphology. Our method enables easy mounting of samples at any angle suited for its morphology. It can be performed with common materials, such as water, clay, and tubes, and if applicable, to samples with a variety of shapes, from a long worm to a round ball.Demonstrating the procedure will be Akiteru Maeno, a technician at the National Institute of Genetics, Japan. To begin, prepare 0.5%agarose by dissolving 500 milligrams of agarose in 100 milliliters of distilled water in a 250-milliliter conical flask, and place the flask in a microwave at the power of 800 watts to heat about one to three minutes. Cool the flask at room temperature.To mount large samples that do not fit in a 1000-microliter micropipette blue tip, such as A.equina, first place the stained sample in a 60-millimeter dish with distilled water to wash off excessive staining solution from the surface. In a 50-milliliter tube, gently pour five milliliters of 0.5%agarose without making bubbles. Place the tube on ice to harden the agarose.Then add 20 milliliters of 0.5%agarose to the tube, place on ice until the agarose begins to harden, and use forceps to place the specimen within the agarose. Adjust the position of the specimen so that its body axis is vertical. Harden the agarose on ice.Place clay on the microCT mounting stage and set the 50-milliliter tube on the clay. To mount small samples that fit in a 1000-microliter micropipette blue tip, such as Harmothoe sp. and X.japonica, decant the stained sample into a 60-millimeter dish without using forceps.With ring tweezers, gently transfer the samples into another 60-millimeter dish filled with distilled water to wash off excessive staining solution from the surface. Next, draw up 100 microliters of 0.5%agarose into a 1000-microliter micropipette blue tip and place it on ice to harden. When handling Harmothoe sp.add 1000 microliters of 0.5%agarose into the agarose plug in the tip. Use ring tweezers to gently transfer Harmothoe sp. from the 60-millimeter dish into the agarose solution in the tip.With a petiolate needle or precision tweezers, adjust the position and orientation of the Harmothoe sp. so that the chaetae are not bent unnaturally. Make sure no bubbles are present, and then place the tip on ice to harden the agarose.When handling fragile samples like X.japonica, use a micropipette to add 1000 microliters of distilled water into the blue tip with the hardened agarose plug. Position the X.Japonica in the distilled water so that it is stable between the walls of the tip. For smaller samples, a 200-microliter micropipette yellow tip can be used.In this case, use 30 microliters of agarose for the plug, and add 200 microliters of agarose or distilled water. Next, cut the tip off a new, 1000-microliter micropipette blue tip, and insert the tip of the plugged tip into the new tip. Place clay on the microCT mounting stage and set the tips with the sample inside on the clay.Promptly after mounting the samples, turn on the x-ray beam at 80 kilovolts and 100 microamps. While observing the x-ray transmission image, move the stage so that the whole sample can be seen by clicking on the x and z-axis buttons, and by manually adjusting the y-axis knob on the mounting stage. Adjust the contrast conditions of the image so that the internal structures can be observed.Adjust the orientation of the sample by changing the angle of the tube in the clay. Rotate the stage 90 degrees by setting the rotation axis to 90, and clicking on the Relative Movement button. Perform the same maneuver four times to complete a full rotation.Manually turn off the x-ray beam each time the sample door is opened, unless the system turns it off automatically. Now click on the z-axis button and manually adjust the y-axis knob to move the mounting stage so that the sample is at the center of the view. Turn the stage by 90 degrees and adjust the sample to the center again.Turn the stage 90 degrees three more times, and adjust as needed to be sure that the sample is at the center of the view from all directions. Next, click on the x-axis button to move the stage along the x-axis toward the x-ray beam source to enlarge the sample, and adjust as needed so that it just fits in the view. Turn the stage and adjust as needed to be sure that the sample fits within the view from all directions.Adjust the scanning conditions and then start scanning. In this experiment, microCT imaging was performed on Actinia equina, Harmothoe sp. and Xenoturbella japonica after staining with 25%Lugol's solution.The staining successfully enhanced the contrast of the internal structures in all specimens, enabling observations of internal soft tissues. This method is applicable to fragile specimens even with external damage, as can be seen from the X.japonica specimen with badly-damaged epidermis. A single high-resolution dataset of a whole specimen was reconstructed for Harmothoe sp.and X.japonica from multiple scans performed on different but overlapping parts of the specimen. Make sure to set the sample in the tube or tip and mount the tube or tip in the microCT so that it does not move during scanning. The data obtained from this procedure can be used for 3-D image reconstructions, enabling the researcher to fully understand both internal and external morphology of the sample.All microCT systems use x-rays, but the specifics may be different from one system to another, so please make sure to closely follow the instructions of each system.

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