Sectioning of Cultured Cells by Ar Ion Beam Milling for SEM Observations
Sectioning of Cultured Cells by Ar Ion Beam Milling for SEM Observations
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通过 Ar 离子束铣削对培养细胞进行切片以进行 SEM 观察
DOI：
10.1017/s1431927620022448
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
S. Tanaka and Y. Ohmi
中科院分区：
文献类型：
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作者：
吉角龍一;長谷川尊之;田中義人;坪田秀平;佐藤井一;S. Tanaka and Y. Ohmi
TEM已被用于通过电子显微镜观察生物样品的横截面。然而，用于TEM观察的样品制备需要费力的程序，并且需要几个小时到一天或更长时间。用于SEM观察的样品制备不太费力，因此SEM越来越多地用于横截面观察。聚焦离子束（FIB）作为一种生物样品的切片技术，由于其在材料科学和工业领域的广泛应用，获得了高精度的切片样品，因此受到了广泛的关注[1]。但是，由于离子束能量高，在生物领域中离子束损伤更为严重，预防措施是非常必要的。在以前的论文[2]中，我们已经报道了一种用于细胞切片的替代技术，该技术基于低能Ar离子束铣削，并且简单快速。在此，我们报告了使用HEK 293 T作为样品获得良好样品的这种方法的细节。在室温下用磷酸盐缓冲盐水（PBS）中的0.1 mg/mL聚-L-赖氨酸包被硅板3 h。用PBS洗涤涂覆的硅板。将HEK 293 T细胞（1.4× 106细胞/ml）与含10%FCS的DMEM培养基一起在聚-L-赖氨酸包被的硅板上于37 ℃下在5%CO2培养箱中培养过夜。然后将硅上的细胞用3%戊二醛溶液固定2 h。用PBS洗涤后，将样品用铂蓝染色。然后通过BEL-1（日新EM）处理样品。在该处理之后，将样品在真空室中干燥。然后将硅板切割成矩形形状。在这个过程中，锋利的边缘是由分裂产生的。锋利的边缘被用作盾牌。最后，将矩形硅片固定到离子切片机（JEOL）的样品保持器上，并照射Ar离子束。离子照射以这样的方式进行，即相对于Ar离子束，大多数细胞处于硅板的阴影中（图1（a））。离子束的直径约为1 mm。在加速电压为1 kV、2kV和3 kV时，离子束电流分别约为20μA、30μA和50μA。在3 kV下进行SEM观察。SEM图像的实例示于图1（B）中。
TEM has been used for cross-sectional observations of biological samples by electron microscopy. However, the sample preparation for TEM observations requires laborious procedures and takes from several hours to a day or more. 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 [1], 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. In a previous paper [2], we have reported an alternative technique for sectioning of cells, which is based on low-energy Ar ion-beam milling, and is easy and rapid. Here, we report the details of this method for obtaining good samples using 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× 10 6 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. 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 of cells is in the shadow of the silicon plate with respect to the Ar ion beam (Fig. 1 (a)). The diameter of the ion beam was about 1mm. The ion beam currents were about 20µA, 30µA and 50µA for accelerating voltages of 1kV, 2kV and 3kV, respectively. SEM observations were performed at 3 kV. An example of the SEM image is shown in Fig. 1 (b).