Konno Alu, Okazaki Shigetoshi
Department of Medical Spectroscopy, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu-City, Shizuoka-Pref 431-3192 Japan.
Zoological Lett. 2018 Jun 6;4:13. doi: 10.1186/s40851-018-0099-6. eCollection 2018.
Investigation of the internal tissues and organs of a macroscopic organism usually requires destructive processes, such as dissection or sectioning. These processes are inevitably associated with the loss of some spatial information. Recently, aqueous-based tissue clearing techniques, which allow whole-organ or even whole-body clearing of small rodents, have been developed and opened a new method of three-dimensional histology. It is expected that these techniques will be useful tools in the field of zoology, in which organisms with highly diverse morphology are investigated and compared. However, most of these new methods are optimized for soft, non-pigmented organs in small rodents, especially the brain, and their applicability to non-model organisms with hard exoskeletons and stronger pigmentation has not been tested.
We explored the possible application of an aqueous-based tissue clearing technique, advanced CUBIC, on small crustaceans. The original CUBIC procedure did not clear the terrestrial isopod, . Therefore, to apply the whole-mount clearing method to isopods with strong pigmentation and calcified exoskeletons, we introduced several pretreatment steps, including decalcification and bleaching. Thereafter, the clearing capacity of the procedure was dramatically improved, and became transparent. The internal organs, such as the digestive tract and male reproductive organs, were visible through sclerites using an ordinary stereomicroscope. We also found that fluorescent nuclear staining using propidium iodide (PI) helped to visualize the internal organs of cleared specimens. Our procedure was also effective on the marine crab, sp.
In this study, we developed a method to clear whole tissues of crustaceans. To the best of our knowledge, this is the first report of whole-mount clearing applied to crustaceans using an aqueous-based technique. This technique could facilitate morphological studies of crustaceans and other organisms with calcified exoskeletons and pigmentation.
对宏观生物体的内部组织和器官进行研究通常需要采用破坏性的方法，如解剖或切片。这些方法不可避免地会导致一些空间信息的丢失。最近，基于水的组织透明化技术已经得到发展，该技术能够实现对小型啮齿动物的全器官甚至全身透明化处理，从而开创了三维组织学的新方法。预计这些技术将成为动物学领域的有用工具，在该领域中，形态高度多样的生物体将得到研究和比较。然而，这些新方法大多是针对小型啮齿动物的柔软、无色素器官（尤其是大脑）进行优化的，其对具有坚硬外骨骼和较强色素沉着的非模式生物的适用性尚未得到测试。
我们探索了一种基于水的组织透明化技术——先进的CUBIC技术在小型甲壳类动物上的可能应用。原始的CUBIC程序无法使陆生等足类动物透明化。因此，为了将整体透明化方法应用于具有强烈色素沉着和钙化外骨骼的等足类动物，我们引入了几个预处理步骤，包括脱钙和漂白。此后，该程序的透明化能力得到了显著提高，并且变得透明。使用普通体视显微镜可以透过硬壳看到内部器官，如消化道和雄性生殖器官。我们还发现，使用碘化丙啶（PI）进行荧光核染色有助于可视化透明化标本的内部器官。我们的程序对海洋蟹也有效。
在本研究中，我们开发了一种使甲壳类动物全组织透明化的方法。据我们所知，这是首次报道使用基于水的技术对甲壳类动物进行整体透明化处理。该技术可以促进对甲壳类动物以及其他具有钙化外骨骼和色素沉着的生物体的形态学研究。
