Aoyama Kazuhiro, Takagi Tomoko, Hirase Ai, Miyazawa Atsuo
FEI Company Japan Ltd., Application Laboratory, NSS-II Building, 2-13-34 Kohnan, Minato-ku, Tokyo 108-0075, Japan.
Ultramicroscopy. 2008 Dec;109(1):70-80. doi: 10.1016/j.ultramic.2008.08.005. Epub 2008 Sep 10.
Scanning transmission electron microscopy (STEM) tomography was applied to biological specimens such as yeast cells, HEK293 cells and primary culture neurons. These cells, which were embedded in a resin, were cut into 1-microm-thick sections. STEM tomography offers several important advantages including: (1) it is effective even for thick specimens, (2) 'dynamic focusing', (3) ease of using an annular dark field (ADF) mode and (4) linear contrasts. It has become evident that STEM tomography offers significant advantages for the observation of thick specimens. By employing STEM tomography, even a 1-microm-thick specimen (which is difficult to observe by conventional transmission electron microscopy (TEM)) was successfully analyzed in three dimensions. The specimen was tilted up to 73 degrees during data acquisition. At a large tilt angle, the specimen thicknesses increase dramatically. In order to observe such thick specimens, we introduced a special small condenser aperture that reduces the collection angle of the STEM probe. The specimen damage caused by the convergent electron beam was expected to be the most serious problem; however, the damage in STEM was actually smaller than that in TEM. In this study, the irradiation damage caused by TEM- and STEM-tomography in biological specimens was quantitatively compared.
扫描透射电子显微镜（STEM）断层扫描技术被应用于酵母细胞、人胚肾293细胞（HEK293细胞）和原代培养神经元等生物样本。这些包埋在树脂中的细胞被切成1微米厚的切片。STEM断层扫描技术具有几个重要优势，包括：（1）即使对于厚样本也有效；（2）“动态聚焦”；（3）易于使用环形暗场（ADF）模式；（4）线性对比度。很明显，STEM断层扫描技术在观察厚样本方面具有显著优势。通过采用STEM断层扫描技术，即使是1微米厚的样本（传统透射电子显微镜（TEM）难以观察）也成功地进行了三维分析。在数据采集过程中，样本倾斜角度达73度。在大倾斜角度下，样本厚度会急剧增加。为了观察如此厚的样本，我们引入了一个特殊的小聚光镜光阑，它减小了STEM探针的收集角度。由会聚电子束引起的样本损伤预计是最严重的问题；然而，实际上STEM中的损伤比TEM中的要小。在本研究中，对生物样本中TEM断层扫描和STEM断层扫描引起的辐照损伤进行了定量比较。
