FIB/SEM tomography with TEM-like resolution for 3D imaging of high-pressure frozen cells
FIB/SEM tomography with TEM-like resolution for 3D imaging of high-pressure frozen cells
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DOI：
10.1007/s00418-012-1020-6
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发表时间：
2012-10-01
影响因子：
2.3
通讯作者：
Walther, Paul
中科院分区：
文献类型：
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作者：
Villinger, Clarissa;Gregorius, Heiko;Walther, Paul
聚焦离子束/扫描电子显微镜 (FIB/SEM) 断层扫描是一种新颖、强大的生物样品三维 (3D) 成像方法。因此，用聚焦离子束（FIB）重复研磨样品，并用扫描电子显微镜（SEM）对每个新产生的块面进行成像。该过程可以任意小增量重复，从而允许对相对较大体积（例如真核细胞）进行 3D 分析。另一方面，高压冷冻和冷冻替代是全细胞电子显微镜制备的黄金标准。在这项工作中，我们结合了这些方法，并通过使用二次电子信号形成图像来显着提高分辨率。通过这种成像模式，在靠近新产生的表面的非常小的、明确的区域中形成对比度。通过使用这种方法，迄今为止只能在透射电子显微镜 (TEM) 中看到的小特征（例如，双层膜的两个小叶、网格蛋白涂层和细胞骨架元素）可以在全细胞 3D 背景下的 FIB/SEM 中直接解析。
Focused ion beam/scanning electron microscopy (FIB/SEM) tomography is a novel powerful approach for three-dimensional (3D) imaging of biological samples. Thereby, a sample is repeatedly milled with the focused ion beam (FIB) and each newly produced block face is imaged with the scanning electron microscope (SEM). This process can be repeated ad libitum in arbitrarily small increments allowing 3D analysis of relatively large volumes such as eukaryotic cells. High-pressure freezing and freeze substitution, on the other hand, are the gold standards for electron microscopic preparation of whole cells. In this work, we combined these methods and substantially improved resolution by using the secondary electron signal for image formation. With this imaging mode, contrast is formed in a very small, well-defined area close to the newly produced surface. By using this approach, small features, so far only visible in transmission electron microscope (TEM) (e.g., the two leaflets of the membrane bi-layer, clathrin coats and cytoskeletal elements), can be resolved directly in the FIB/SEM in the 3D context of whole cells.