Hamano Tohru, Dwiranti Astari, Kaneyoshi Kohei, Fukuda Shota, Kometani Reo, Nakao Masayuki, Takata Hideaki, Uchiyama Susumu, Ohmido Nobuko, Fukui Kiichi
1Laboratory of Dynamic Cell Biology,Department of Biotechnology,Graduate School of Engineering,Osaka University,Yamadaoka,Suita,Osaka 565-0871,Japan.
2Laboratory of Nano Mechanics,Department of Mechanical Engineering,Graduate School of Engineering,The University of Tokyo,Hongo,Bunkyo,Tokyo 113-8685,Japan.
Microsc Microanal. 2014 Oct;20(5):1340-7. doi: 10.1017/S143192761401280X. Epub 2014 Jul 10.
Attempts to elucidate chromosome structure have long remained elusive. Electron microscopy is useful for chromosome structure research because of its high resolution and magnification. However, biological samples such as chromosomes need to be subjected to various preparation steps, including dehydration, drying, and metal/carbon coating, which may induce shrinkage and artifacts. The ionic liquid technique has recently been developed and it enables sample preparation without dehydration, drying, or coating, providing a sample that is closer to the native condition. Concurrently, focused ion beam/scanning electron microscopy (FIB/SEM) has been developed, allowing the investigation and direct analysis of chromosome interiors. In this study, we investigated chromosome interiors by FIB/SEM using plant and human chromosomes prepared by the ionic liquid technique. As a result, two types of chromosomes, with and without cavities, were visualized, both for barley and human chromosomes prepared by critical point drying. However, chromosome interiors were revealed only as a solid structure, lacking cavities, when prepared by the ionic liquid technique. Our results suggest that the existence and size of cavities depend on the preparation procedures. We conclude that combination of the ionic liquid technique and FIB/SEM is a powerful tool for chromosome study.
长期以来，阐明染色体结构的尝试一直难以实现。电子显微镜因其高分辨率和放大倍数，对染色体结构研究很有用。然而，像染色体这样的生物样本需要经过各种制备步骤，包括脱水、干燥和金属/碳涂层处理，这些步骤可能会导致样本收缩和产生假象。离子液体技术最近得到了发展，它能够在不进行脱水、干燥或涂层处理的情况下制备样本，从而提供更接近原始状态的样本。与此同时，聚焦离子束/扫描电子显微镜（FIB/SEM）也已得到发展，使得对染色体内部结构的研究和直接分析成为可能。在本研究中，我们使用离子液体技术制备的植物和人类染色体，通过FIB/SEM对染色体内部结构进行了研究。结果发现，对于通过临界点干燥制备的大麦和人类染色体，都观察到了有腔和无腔两种类型的染色体。然而，当使用离子液体技术制备时，染色体内部仅呈现为缺乏腔的固体结构。我们的结果表明，腔的存在和大小取决于制备程序。我们得出结论，离子液体技术和FIB/SEM的结合是染色体研究的有力工具。
