3D imaging of cells and tissues by focused ion beam/scanning electron microscopy (FIB/SEM).
3D imaging of cells and tissues by focused ion beam/scanning electron microscopy (FIB/SEM).
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DOI：
10.1007/978-1-62703-137-0_16
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发表时间：
2013-01-01
期刊：
影响因子：
--
通讯作者：
Drobne, Damjana
中科院分区：
文献类型：
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作者：
Drobne, Damjana
将扫描电子显微镜（SEM）和聚焦离子束（FIB）技术集成到一个单一的FIB/SEM系统中，允许使用FIB作为纳米手术刀来揭示特定部位的地下微结构，这些微结构可以通过SEM进行非常详细的检查。FIB/SEM技术在半导体工业和材料科学中有着广泛的应用，近年来在生命科学领域的应用已经开始。用于FIB/SEM研究的样品可以嵌入塑料基质中，这是制备透射电子显微镜（TEM）样品的传统方法，也可以像制备SEM成像的样品一样简单地干燥。目前，FIB/SEM在生命科学中被用于(a)制备用于TEM分析的薄片的提升技术，(b)嵌入在基质中的样品的断层扫描，以及(c)使用大范围放大的原位特定位置的FIB铣削和SEM成像。位点特异性铣削和成像技术作为一种研究单个真核和原核细胞、小动物和不同动物组织结构的技术引起了广泛的兴趣，但仍有待更深入的探索。过去，通过FIB/SEM制备特定部位的铣削和成像样品通常采用用于TEM样品的包埋技术，这在文献中已经得到了很好的描述。在FIB/SEM中使用干燥样品的样品制备方案的研究较少。本章的目的是鼓励FIB/SEM在干燥生物样品上的应用。详细介绍了常规干燥样品制备和干燥生物样品的FIB/SEM研究。描述和说明了重要的步骤，并提供了相同组织的包埋和干燥样品之间的直接比较。在纳米尺度上发现组织或器官的大体形态、任何选定区域的表面特征和细胞内结构细节之间的联系的能力是电子显微镜在生命科学中的一个有吸引力的应用，值得进一步探索。
Integration of a scanning electron microscope (SEM) and focused ion beam (FIB) technology into a single FIB/SEM system permits use of the FIB as a nano-scalpel to reveal site-specific subsurface microstructures which can be examined in great detail by SEM. The FIB/SEM technology is widely used in the semiconductor industry and material sciences, and recently its use in the life sciences has been initiated. Samples for FIB/SEM investigation can be either embedded in a plastic matrix, the traditional means of preparation of transmission electron microscopy (TEM) specimens, or simply dried as in samples prepared for SEM imaging. Currently, FIB/SEM is used in the life sciences for (a) preparation by the lift-out technique of lamella for TEM analysis, (b) tomography of samples embedded in a matrix, and (c) in situ site-specific FIB milling and SEM imaging using a wide range of magnifications. Site-specific milling and imaging has attracted wide interest as a technique in structural research of single eukaryotic and prokaryotic cells, small animals, and different animal tissue, but it still remains to be explored more thoroughly. In the past, preparation of samples for site-specific milling and imaging by FIB/SEM has typically adopted the embedding techniques used for TEM samples, and which have been very well described in the literature. Sample preparation protocols for the use of dried samples in FIB/SEM have been less well investigated. The aim of this chapter is to encourage application of FIB/SEM on dried biological samples. A detailed description of conventional dried sample preparation and FIB/SEM investigation of dried biological samples is presented. The important steps are described and illustrated, and direct comparison between embedded and dried samples of same tissues is provided. The ability to discover links between gross morphology of the tissue or organ, surface characteristics of any selected region, and intracellular structural details on the nanometer scale is an appealing application of electron microscopy in the life sciences and merits further exploration.