Heymann Jurgen A W, Hayles Mike, Gestmann Ingo, Giannuzzi Lucille A, Lich Ben, Subramaniam Sriram
Laboratory of Cell Biology, National Cancer Institute, NIH, Bethesda, MD, USA.
J Struct Biol. 2006 Jul;155(1):63-73. doi: 10.1016/j.jsb.2006.03.006. Epub 2006 Apr 4.
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.
当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三维成像的方法虽然强大，但仅限于相对薄且透明的标本。在此，我们报告了一种新一代双束电子显微镜的应用，该显微镜能够在空间分辨率上比目前光学显微镜所达到的分辨率高约一个数量级的情况下，对细胞和组织标本内部进行特定部位成像。成像原理基于使用聚焦离子束在标本的指定部位进行切割，然后用扫描电子束观察新生成的表面。这两个步骤重复几次，就会生成一系列以规则间隔排列的标本表面图，这些图可以转换为标本的三维图。我们已经探索了这种顺序“切片并观察”策略在冷冻酵母细胞和肿瘤组织特定部位三维成像中的潜力，并确定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。我们还表明，使用离子束对树脂包埋的标本进行“铣削”可以原位生成200纳米厚的切片，这为使用超薄切片机手动切割细胞和组织提供了一种有价值的替代方法。我们的结果表明，双束成像对于基础生物医学和临床应用中的细胞和亚细胞三维成像而言是一种强大的新工具。
