Application of Ar Ion Beam Milling on Sectioning of Cells for SEM Observations
Application of Ar Ion Beam Milling on Sectioning of Cells for SEM Observations
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Ar 离子束铣削在用于 SEM 观察的细胞切片中的应用
DOI：
10.1017/s1431927619005245
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Yusuke Ohmi
中科院分区：
文献类型：
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作者：
Shigeyasu Tanaka;Yusuke Ohmi
用电子显微镜对生物样品进行横断面观察时，通常要用到透射电子显微镜。然而，用于瞬变显微镜观测的样品制备需要费力的程序，需要几个小时到一天或更长时间[1]。扫描电子显微镜观察的样品制备不那么费力，因此越来越多地使用扫描电子显微镜进行横断面观察。聚焦离子束(FIB)作为一种生物样品的切片技术，因其在材料科学和工业领域的应用而备受关注[2]，用于获得精确的切片样品。但是，由于离子束能量较高，在生物领域引起的损伤更为严重，因此必须采取预防措施。我们开发了一种替代的细胞切片技术，该技术基于低能Ar离子束球磨，简单、快速。以HEK293T细胞为研究对象，用0.1 mg/mL聚L赖氨酸在磷酸盐缓冲液(PBS)中包被硅片，室温下包膜3h。镀膜后的硅板用PBS清洗。将HEK293T细胞(1.4×106cell/ml)接种于含10%胎牛血清的DMEM培养液中，37℃，5%CO2培养箱中过夜。然后将细胞固定在硅片上，用3%的戊二醛溶液固定2 h，用PBS洗涤后，用铂蓝染色。标本经BEL-1(Nisshin EM)处理。经过这一处理后，样品在真空室中干燥。然后将硅板切割成矩形(约0.5 mm×3 mm)。在这个过程中，通过解理产生了锋利的边缘。锋利的边缘被用作盾牌。最后，将矩形硅片固定在离子切片机(JEOL)的样品固定器上，并对Ar离子束进行辐照。离子辐照是以这样的方式进行的，即大多数电池位于相对于Ar离子束的硅板的阴影中(图1)。只有靠近硅板边缘的电池的上部被溅射。可以通过改变片材表面与Ar离子束之间的角度θ来控制分割的位置。通过这种方法，可以很容易地制备切片样品。扫描电子显微镜观察是在3KV的加速电压下进行的。
For cross-sectional observations of biological samples by electron microscopy, TEM is usually used. However, the sample preparation for TEM observations requires laborious procedures and takes from several hours to a day or more [1]. The sample preparation for SEM observations is less laborious, thus SEM is used more and more for cross-sectional observations. As a sectioning technique for biological samples, focused ion beam (FIB) has received much attention [2], because it has been used in material science and industry fields to obtain accurately and precisely sectioned samples. But, because of the high beam energy, ion-beam induced damage is more severe in the biological field, and precautions are vitally necessary. We developed an alternative technique for sectioning of cells, which is based on lowenergy Ar ion-beam milling, and is easy and rapid. Here, we report our method using a cultured cell (HEK293T) as a sample.A silicon plate was coated by 0.1 mg/mL poly-L-lysine in phosphate buffered saline (PBS) for 3 h at room temperature. The coated silicon plate was washed by PBS. HEK293T cell (1.4× 106 cell/ml) was cultured with 10% FCS containing DMEM medium on the poly-L-lysine coated silicon plate overnight at 37ºC in 5% CO2 incubator. Then the cell on silicon was fixed with 3% glutaraldehyde solution for 2 h. After washing with PBS, the sample was stained with Platinum Blue. Then the sample was processed by BEL-1 (Nisshin EM). After this treatment, the sample was dried in a vacuum chamber. Then the silicon plate was cut into a rectangular shape (about 0.5 mm× 3 mm). In this process, sharp edges were created by cleavage. The sharp edge was used as a shield. Finally, the rectangular silicon piece was fixed to a sample holder of Ion Slicer (JEOL), and Ar ion beam was irradiated. The ion irradiation was carried out in such a way that most cells were located in the shadow of the silicon plate with respect to the Ar ion beam (Figure 1). Only upper portion of the cells near the edge of the silicon plate is sputtered. The position of sectioning can be controlled by changing the angle θ between the surface of the piece and the Ar ion beam. In this way, sectioned samples can be prepared easily. SEM observations were performed at an accelerating voltage of 3 kV.