From 2D slices to 3D volumes: Image based reconstruction and morphological characterization of hippocampal cells on charged and uncharged surfaces using FIB/SEM serial sectioning
From 2D slices to 3D volumes: Image based reconstruction and morphological characterization of hippocampal cells on charged and uncharged surfaces using FIB/SEM serial sectioning
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DOI：
10.1016/j.ultramic.2010.12.017
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发表时间：
2011-03-01
期刊：
影响因子：
2.2
通讯作者：
Zehbe, Rolf
中科院分区：
文献类型：
--
作者：
Schmidt, Franziska;Kuehbacher, Markus;Zehbe, Rolf
亚细胞分辨率的3D成像是生命科学中研究细胞及其与天然组织或人造物体相互作用的强大工具。虽然可以用X射线或电子建立实现足够的结构分辨率的层析成像实验设置，但由于电子的穿透深度差，电子的使用通常限于透射电子显微镜中非常薄的样品。因此，在最先进的双束实验装置中，连续切片方法和扫描电子显微镜的结合提供了一种使用聚焦离子束进行切片和电子束进行成像来对高分辨率空间细节进行成像的方法。这种技术优于X射线μ CT或X射线显微镜的优点在于，吸收不是成像中的限制因素，因此即使是强吸收结构也可以以更高的可能分辨率进行空间重建。在这项研究中使用这种方法来阐明从海马细胞系（HT 22）沉积在金微电极上的细胞的形态上的电位的影响。虽然在两种不同对照（金和聚合物基底）上培养的细胞确实显示出预期的拉伸形态，但阳极和阴极上的细胞显著不同。沉积在电极阳极部分的细胞表现出最极端的偏差，几乎呈球形，并表现出染色质凝聚的迹象，可能表明细胞死亡。此外，EDX被用作化学和结构分析相结合的补充方法。(C)2010 Elsevier B.V.保留所有权利。
3D imaging at a subcellular resolution is a powerful tool in the life sciences to investigate cells and their interactions with native tissues or artificial objects. While a tomographic experimental setup achieving a sufficient structural resolution can be established with either X-rays or electrons, the use of electrons is usually limited to very thin samples in transmission electron microscopy due to the poor penetration depths of electrons. The combination of a serial sectioning approach and scanning electron microscopy in state of the art dual beam experimental setups therefore offers a means to image highly resolved spatial details using a focused ion beam for slicing and an electron beam for imaging. The advantage of this technique over X-ray mu CT or X-ray microscopy attributes to the fact that absorption is not a limiting factor in imaging and therefore even strong absorbing structures can be spatially reconstructed with a much higher possible resolution. This approach was used in this study to elucidate the effect of an electric potential on the morphology of cells from a hippocampal cell line (HT22) deposited on gold microelectrodes. While cells cultivated on two different controls (gold and polymer substrates) did show the expected stretched morphology, cells on both the anode and the cathode differed significantly. Cells deposited on the anode part of the electrode exhibited the most extreme deviation, being almost spherical and showed signs of chromatin condensation possibly indicating cell death. Furthermore, EDX was used as supplemental methodology for combined chemical and structural analyses. (C) 2010 Elsevier B.V. All rights reserved.