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Environnement

微聚焦 X 射线 CT (微CT) 阿克蒂尼亚 equina (Cnidaria),哈莫特霍sp. (安妮利达) 和Xenoturbella japonica (西纳科洛莫卡) 成像

Published: August 06, 2019
doi:
10.3791/59161
, , ,
1Mammalian Genetics Laboratory,National Institute of Genetics, 2Misaki Marine Biological Station,The University of Tokyo, 3Shimoda Marine Research Center,University of Tsukuba

Summary

在这里,详细介绍了对三种海洋无脊椎动物进行微聚焦X射线计算机断层成像(微CT)成像的协议。本研究描述了样品固定、染色、安装、扫描、图像重建和数据分析等步骤。还就如何针对不同的样本调整协议提出了建议。

Abstract

传统上,生物学家不得不依靠破坏性方法,如切片,以调查不透明生物的内部结构。非破坏性微焦 X 射线计算机断层扫描 (microCT) 成像已成为生物学中一个强大而新兴的协议,这是由于样品染色方法的技术进步以及微CT硬件、处理计算机和数据的创新分析软件。然而,该协议并不普遍使用,因为它是在医疗和工业领域。这种有限使用的原因之一是缺乏一个简单而易于理解的手册,涵盖了所有必要的步骤:样品收集、固定、染色、安装、扫描和数据分析。另一个原因是元类动物,特别是海洋无脊椎动物的多样化。由于海洋无脊椎动物的尺寸、形态和生理条件各不相同,因此根据样品,在每一步调整实验条件和硬件配置至关重要。在这里,微CT成像方法使用三种植物遗传多样性的海洋无脊椎动物详细解释:Actinia equina(安索佐亚,尼达里亚),哈莫特霍sp.(波利查塔,安妮利达),和Xenoturbella japonica((西诺图贝利达,西纳科洛莫法)。提出了对各种动物进行微CT成像的建议。

Introduction

生物研究人员通常不得不进行薄截面,并通过光或电子显微镜进行观察,以调查不透明生物体的内部结构。然而,这些方法是破坏性的,当应用于稀有或有价值的标本时,是破坏性的和有问题的。此外,该方法中的几个步骤(如嵌入和切片)非常耗时,并且可能需要几天时间才能观察样本,具体取决于协议。此外,在处理许多部分时,始终有可能损坏或丢失某些部分。组织清除技术可用于一些标本1,2,3,4,5,但还不适用于许多动物物种。

为了克服这些问题,一些生物学家已经开始使用微焦X射线计算机断层扫描(微CT)成像6,7,8,9,10,11, 12,13,14,15.在 X 射线 CT 中,标本从围绕样品移动的 X 射线源产生不同角度的 X 射线进行照射,并且传输的 X 射线由探测器进行监测,该探测器也在样品周围移动。对获得的X射线传输数据进行了分析,以重建标本的横截面图像。此方法可以观察内部结构,而不会破坏样品。由于其安全性和方便性,它常用于医疗和牙科应用,CT 系统可在全球的医院和牙科中心找到。此外,工业X射线CT还经常用于观察工业领域的非医学样品,用于检查和计量。与X射线源和探测器移动的医学CT不同,这两个部分固定在工业CT中,样品在扫描过程中旋转。工业CT通常比医疗CT产生更高的分辨率图像,被称为微CT(微米级分辨率)或纳米CT(纳米级分辨率)。最近,使用微CT的研究在生物学的各个领域迅速增加,14,15,16,17,18,1920,21,22,23,24,25,26,27,28,29,30,31,32,33,34.

使用CT的生物研究最初针对内部结构,主要由硬组织,如骨。使用各种化学剂染色技术的进步使各种生物体中的软组织可视化6、7、8、9、14、15 ,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34.在这些试剂中,碘基造影剂相对安全、价格低廉,可用于各种生物体中软组织的可视化7、14。关于海洋无脊椎动物,微CT已广泛应用于软体动物,如软体动物6,25,32,33,安妮利德18,19,20,28,和阿特霍波德21,23,29,31 。然而,很少有关于其他动物的报导,如布约佐斯6,异种动物26,和仙人掌24,30。一般来说,对海洋无脊椎动物使用微CT的研究比对脊椎动物的研究要少。这种对海洋无脊椎动物的有限使用,一个主要原因是这些动物观察到的巨大多样性。由于其不同的大小、形态和生理,每个物种对不同的实验程序有不同的反应。因此,在样品制备过程中,选择最合适的固定和染色试剂,并为每个物种调整每个步骤设置条件至关重要。同样,还需要为每个样品设置适当的扫描配置,如安装方法、电压、电流、机械放大速率和空间分辨率功率。为了克服这个问题,一份简单而易懂的手册涵盖了所有必要的步骤,解释了如何根据样本调整每个步骤,并展示来自多个样本的详细示例至关重要。

在本研究中,我们使用三种海洋无脊椎动物物种,逐步描述了从样品固定到数据分析的微CT成像方案。在东京大学Misaki海洋生物站附近采集了海葵阿基尼亚埃奎纳(安佐佐亚,Cnidaria)的标本。他们有一个球形,柔软的身体,直径约2厘米(图1A-C)。哈莫特霍(波利查塔,安妮利达)样本也在米萨基海洋生物站附近采集。它们是长约1.5厘米的细长蠕虫,整个身体都存在坚韧的沙塔(图1D)。第13次JAMBIO海岸生物联合调查期间,在筑波大学Shimoda海洋研究中心附近采集了一个Xenoturbella japonica35(西诺韦利达,西纳科洛莫卡)标本。它是一种软体蠕虫,长约0.8厘米(图1E)。详细说明了对每个样品的条件和配置所做的调整。我们的研究就如何对海洋无脊椎动物进行微CT成像提供了几点建议,我们希望这将激励生物学家利用这个方案进行研究。

Protocol

1. 固定 对于Actinia equina,在室温下,在10%MgCl2海水中放松动物约15分钟。转移到70%乙醇,并在室温下储存。 对于Harmothoe sp.,通过在冰冷的海水中放置动物进行约15分钟的麻醉,将10%(v/v)正式溶液与海水一起转移到,并在室温下储存。 对于异位肌醇酶,在淡水中使用7%的MgCl2来放松动物。在过滤的海水中过夜4%的甲醛(PFA)。放入70%乙醇,储存在4°C。注?…

Representative Results

在用25%的卢戈尔溶液染色样品后,我们对A.equina(安索佐亚、尼尼达)、哈莫特霍(波利查塔、安妮利达)和X.japonica(西诺韦利达,西纳科洛莫卡)进行了微CT成像。 染色成功地增强了所有标本内部结构的对比度,从而能够观察内部软组织(图6)。连同过去的报告6,7,16,19,<su…

Discussion

使用形式辛的固定剂,如本研究中使用的海水中的10%(v/v)形式化溶液,已知能保存各种海洋无脊椎动物的形态,并常用于微CT成像18、24、25 ,26,28,30,33.然而,近年来,一些国家对使用这种化学品的限制变得严格,可以使用甲醛或谷醛?…

Divulgations

The authors have nothing to disclose.

Acknowledgements

我们要感谢石破茂在研究期间给予的帮助和提供的研究环境。我们感谢Yanagi和TakatoIzumi就A.equina和Masaatsu Tanaka提供关于Harmothoe sp.标本的建议。我们要感谢筑波大学石田海洋研究中心和东京大学三崎海洋生物站的工作人员在样品采集方面的帮助。我们要感谢编辑(www.editage.jp)的英语语言编辑。这项工作得到了日本青年科学家援助资助组织(JP26711022)和日本海洋生物协会JAMBIO的支持。

Materials

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
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Tags

MicroCTActinia EquinaHarmothoe Sp.Xenoturbella JaponicaMarine InvertebratesSample MountingAgaroseStainingSample Preparation
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Maeno, A., Kohtsuka, H., Takatani, K., Nakano, H. Microfocus X-ray CT (microCT) Imaging of Actinia equina (Cnidaria), Harmothoe sp. (Annelida), and Xenoturbella japonica (Xenacoelomorpha). J. Vis. Exp. (150), e59161, doi:10.3791/59161 (2019).
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