Kang Byung-Ho
School of Life Sciences, Centre for Cell and Developmental Biology and State Key Laboratory of Agrobiotechnology, Chinese University of Hong Kong, Hong Kong, China.
Methods Mol Biol. 2016;1496:55-62. doi: 10.1007/978-1-4939-6463-5_5.
Because of the weak penetrating power of electrons, the signal-to-noise ratio of a transmission electron micrograph (TEM) worsens as section thickness increases. This problem is alleviated by the use of the scanning transmission electron microscopy (STEM). Tomography analyses using STEM of thick sections from yeast and mammalian cells are of higher quality than are bright-field (BF) images. In this study, we compared regular BF tomograms and STEM tomograms from 500-nm thick sections from hypertrophied Golgi stacks of alfalfa root cap cells. Due to their thickness and intense heavy metal staining, BF tomograms of the thick sections suffer from poor contrast and high noise levels. We were able to mitigate these drawbacks by using STEM tomography. When we performed STEM tomography of densely stained chloroplasts of Arabidopsis cotyledon, we observed similar improvements relative to BF tomograms. A longer time is required to collect a STEM tilt series than similar BF TEM images, and dynamic autofocusing required for STEM imaging often fails at high tilt angles. Despite these limitations, STEM tomography is a powerful method for analyzing structures of large or dense organelles of plant cells.
由于电子的穿透能力较弱，随着切片厚度增加，透射电子显微镜图像（TEM）的信噪比会变差。扫描透射电子显微镜（STEM）的使用缓解了这一问题。使用STEM对酵母和哺乳动物细胞的厚切片进行断层扫描分析，其质量高于明场（BF）图像。在本研究中，我们比较了苜蓿根冠细胞肥大高尔基体堆叠500纳米厚切片的常规BF断层图像和STEM断层图像。由于切片厚度和强烈的重金属染色，厚切片的BF断层图像对比度差且噪声水平高。我们通过使用STEM断层扫描减轻了这些缺点。当我们对拟南芥子叶中密集染色的叶绿体进行STEM断层扫描时，相对于BF断层图像，我们观察到了类似的改善。收集STEM倾斜序列所需的时间比类似的BF TEM图像更长，并且STEM成像所需的动态自动聚焦在高倾斜角度时经常失败。尽管存在这些局限性，STEM断层扫描仍是分析植物细胞大型或密集细胞器结构的有力方法。
