Suzuki Michiyo, Sakashita Tetsuya, Funayama Tomoo
Department of Radiation-Applied Biology Research, Takasaki Advanced Radiation Research Institute, National Institutes for Quantum and Radiological Science and Technology (QST-Takasaki);
Department of Radiation-Applied Biology Research, Takasaki Advanced Radiation Research Institute, National Institutes for Quantum and Radiological Science and Technology (QST-Takasaki).
J Vis Exp. 2019 Mar 19(145). doi: 10.3791/59008.
Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 µm thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.
辐射广泛应用于生物学领域和离子束育种，在这些方法中，微束辐照是识别活生物体中辐射敏感位点的有力手段。本文描述了一系列为秀丽隐杆线虫活体进行靶向微束辐照而开发的芯片上固定方法。值得注意的是，详细解释了我们之前开发的用于固定秀丽隐杆线虫个体而无需麻醉的聚二甲基硅氧烷（PDMS）微流控芯片的处理方法。这种芯片称为虫片，具有弹性，能使微流控通道扩张，其弹性可使动物被轻柔包裹。此外，由于PDMS的自吸附能力，通过用薄覆盖膜覆盖虫片表面，可将动物密封在通道中，动物不会被推入通道进行封闭。翻转覆盖膜，我们就能轻松收集动物。此外，虫片具有保水性，可使秀丽隐杆线虫个体在活体条件下长时间进行显微镜观察。另外，该片仅300微米厚，能让碳离子等重离子穿过包裹动物的片子，从而准确检测离子粒子并测量所施加的辐射剂量。由于选择用于封闭动物的覆盖膜对成功进行长期固定非常重要，我们进行了合适覆盖膜的选择，并在一些膜中展示了推荐的一种。作为芯片的应用示例，我们介绍了对包围虫片微流控通道的动物肌肉活动的成像观察以及微束辐照。这些示例表明虫片极大地扩展了生物学实验的可能性。
