Site-specific 3D imaging of cells and tissues with a dual beam microscope
Site-specific 3D imaging of cells and tissues with a dual beam microscope
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DOI：
10.1016/j.jsb.2006.03.006
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发表时间：
2006-07-01
影响因子：
3
通讯作者：
Subramaniam, Sriram
中科院分区：
文献类型：
--
作者：
Heymann, Jurgen A. W.;Hayles, Mike;Subramaniam, Sriram
目前使用共聚焦显微镜和电子断层扫描在亚细胞分辨率下进行3D成像的方法虽然功能强大，但仅限于相对较薄和透明的样品。在这里，我们报告了新一代双光束电子显微镜的使用，该显微镜能够以比目前光学显微镜更好的数量级的空间分辨率对细胞和组织标本的内部进行特定部位的成像。成像的原理是使用聚焦的离子束在样品中的指定位置创建一个切割，然后用扫描电子束观察新生成的表面。因此，这两个步骤的多次迭代导致以规则间隔产生一系列样品的表面图，其可被转换为样品的三维图。我们已经探索了这种连续的“切片和观察”策略用于冷冻酵母细胞和肿瘤组织的部位特异性3D成像的潜力，并建立了这种方法可以识别细胞内特征的位置，例如100纳米宽的酵母核孔复合体。我们还显示，200纳米厚的切片可以通过使用离子束对树脂包埋的样品进行原位“球磨”来生成，这提供了一种使用超显微切片机对细胞和组织进行手动切片的有价值的替代方案。我们的结果表明，双束成像是一种强大的细胞和亚细胞三维成像工具，在基础生物医学和临床应用中都是如此。(C)2006 Elsevier Inc.保留所有权利。
Current approaches to 3D imaging at subcellular resolution using confocal microscopy and electron tomography, while powerful, are limited to relatively thin and transparent specimens. Here we report on the use of a new generation of dual beam electron microscopes capable of site-specific imaging of the interior of cellular and tissue specimens at spatial resolutions about an order of magnitude better than those currently achieved with optical microscopy. The principle of imaging is based on using a focused ion beam to create a cut at a designated site in the specimen, followed by viewing the newly generated surface with a scanning electron beam. Iteration of these two steps several times thus results in the generation of a series of surface maps of the specimen at regularly spaced intervals, which can be converted into a three-dimensional map of the specimen. We have explored the potential of this sequential "slice-and-view" strategy for site-specific 3D imaging of frozen yeast cells and tumor tissue, and establish that this approach can identify the locations of intracellular features such as the 100 nm-wide yeast nuclear pore complex. We also show that 200 nm thick sections can be generated in situ by "milling" of resin-embedded specimens using the ion beam, providing a valuable alternative to manual sectioning of cells and tissues using an ultramicrotome. Our results demonstrate that dual beam imaging is a powerful new tool for cellular and subcellular imaging in 3D for both basic biomedical and clinical applications. (c) 2006 Elsevier Inc. All rights reserved.