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

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.