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

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 specimens^1,

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.....

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 reports^6,7,

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 imaging^{18,24,25,26,28,30,33}. However, restrictions on the use of this chemical have become str.......

Materials

List of materials used in this article

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