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Method Article

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

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DOI:

10.3791/59161

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August 6th, 2019

 ,  ,  , 
1Mammalian Genetics Laboratory, National Institute of Genetics, 2Misaki Marine Biological Station, The University of Tokyo, 3Shimoda Marine Research Center, University of Tsukuba

Corresponding Authors: Hiroaki Nakano <h.nakano@shimoda.tsukuba.ac.jp>

In This Article

Summary

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.

Abstract

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’ 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.

Introduction

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 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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Protocol

1. Fixation

  1. 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.
  2. 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.
  3. 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 °C.
    CAUTION: PFA is hazardous and must be handled with care.

2. Staining

  1. 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.
  2. Replace the solution with distilled water (DW) and store the samples in DW at room temperature for 2 h. Repeat this step three times.
  3. 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.
  4. Decant the DW from the samples and pour in the 25% Lugol solution. Stain for 24 h at room temperature.

3. Stage Mounting

  1. 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 °C by keeping at room temperature.
    CAUTION: Do not overheat or completely seal the flask when heating to prevent the agarose from boiling over.
  2. Mount large 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 μL micropipette 'blue' tip.
    1. Place the stained sample in a 60-mm dish with DW to wash off excessive staining solution from the surface.
    2. 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.
    3. 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.
    4. Adjust the position and orientation of the sample with forceps and harden the agarose on ice.
    5. Place clay on the microCT mounting stage and set the 50 mL tube on the clay (Figure 2A).
  3. Mount small samples such as Harmothoe sp. and X. japonica using a 1,000 µL micropipette 'blue' tip.
    NOTE: For smaller samples, 200 μL micropipette ‘yellow’ tip can be used. In this case, use 30 μL of agarose for the plug and add 200 μL of agarose or distilled water.
    1. Draw up 100 µL of 0.5% agarose into a 1,000 µL micropipette 'blue' tip and harden the agarose by holding the tip over ice, making a plug in the tip (Figure 2B-a).
    2. Decant the stained sample into a 60-mm dish without using forceps.
    3. Gently transfer the sample using ring tweezers into another 60-mm dish with DW to wash off excessive staining solution from the surface.
    4. Add 1,000 µL of either 0.5% agarose or 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.
    5. Gently transfer the sample from the 60-mm dish into the agarose or DW in the plugged tip using ring tweezers.
    6. 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.
    7. Cut the tip off a new 1,000 µL micropipette 'blue' tip (Figure 2B-b,c) and insert the tip of the plugged tip into the new tip.
    8. 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.

4. MicroCT scanning

  1. Turn on the X-ray beam at 80 kV, 100 µA.
  2. 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).
  3. Adjust the orientation of the sample by changing the angle of the tube/tip in the clay (Figure 2A). Rotate the stage 90° 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.
  4. 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° and do the same. Turn the stage 360° and check that the sample is at the center of the view from all directions.
  5. 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).
  6. Turn the stage 360° and adjust as needed so that the sample fits within view from all directions.
  7. Adjust the scanning conditions as shown in Table 1.
  8. Start scanning; it takes about 10 min.

5. Image reconstruction

  1. Start up the microCT system's accessory software (see the Table of Materials) and open the scanned data.
  2. Adjust differences in the rotation axis of the sample during scanning by clicking on the automatic shift value calculation button (Figure 4A: green box).
  3. Adjust the orientation of the image by rotating the orange arrows (Figure 4B). If the orientation was changed, repeat step 5.2.
  4. Click on the area tab (Figure 4C: magenta box) and trim areas where samples are not present (Figure 4C: yellow box).
  5. 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.
  6. Perform reconfiguration by clicking on the reconfiguration button (Figure 4D: green box).
  7. Adjust image brightness and contrast by setting the black and white values as black value 0, white value 250 (Figure 4D: blue box).
  8. 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 (µm)_number.tiff.
    NOTE: The original microCT datasets from this study are available in the Figshare repository, doi:10.6084/m9.figshare.767083736.

6. Data analyses

  1. 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.
  2. Click on the import icon (Figure 5C: magenta box), select the dataset saved in section 5, and click open.
  3. Click on the copy links button (Figure 5D) to import the data.
  4. Display the 2D cross-section by clicking on the 2D viewer icon (Figure 5C: blue box).
  5. 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]).
  6. 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).
  7. Check other cross-section images by moving the scrollbar (Figure 5G: box).
  8. 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).
  9. Click on the displayed image, and choose the export tab to store cross-section images.

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Results

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,

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Discussion

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...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
250-ml Erlenmeyer flaskCorningCLS430183
5-ml Sampling tube ST-500BIO-BIK103010
50-ml Polypropylene tubeGreiner Bio One International227261
60-mm Non-treated DishIWAKI1010-060
AgarosePromegaV3125
Ecological grade tip (blue) 1000 µlBMBioBIO1000RF
EthanolWako Pure Chemical Industries057-00451
FormalinWako Pure Chemical Industries061-00416
IodineWako Pure Chemical Industries094-05421
Magnesium chloride hexahydrateWako Pure Chemical Industries135-00165
OsiriX DICOM ViewerPixmeo SARLOsiriX MD v10.0https://www.osirix-viewer.com
ParaformaldehydeWako Pure Chemical Industries163-25983
Petiolate needleAS ONE2-013-01
Pipetman P200 MicropipetteGILSONF123601
Pipetman P1000 MicropipetteGILSONF123602
Potassium iodideWako Pure Chemical Industries166-03971
Precision tweezers 5DUMONT0302-5-PS
QuickRack MultI fit tip (yellow) 200 ulSorenson10660
Razor bladesFeatherFA-10
Ring tweezersNAPOXA-26
Stereoscopic microscopeLeicaMZ95
X-ray Micro-CT imaging systemComscantechnoScanXmate-E090S105

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