Cryo FIB-SEM: Volume imaging of cellular ultrastructure in native frozen specimens
Cryo FIB-SEM: Volume imaging of cellular ultrastructure in native frozen specimens
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DOI：
10.1016/j.jsb.2013.09.024
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发表时间：
2013-11-01
影响因子：
3
通讯作者：
Moebius, Wiebke
中科院分区：
文献类型：
--
作者：
Schertel, Andreas;Snaidero, Nicolas;Moebius, Wiebke
越来越需要高分辨率的体积显微镜来更好地理解三维组装体中的细胞功能。聚焦离子束（FIB）铣削用于扫描电子显微镜（SEM）中的连续块面成像是一种有效且快速的方法来生成用于3D分析的此类体积数据。在这里，我们应用这种技术在低温条件下，图像完全水合冷冻标本的小鼠视神经和枯草芽孢杆菌孢子获得高压冷冻（HPF）。我们建立了成像条件，通过使用透镜内二次电子（SE）检测器在-150 ℃下直接观察块面的超微结构。通过使用聚焦离子束进行连续切片并对视神经进行块面成像，我们获得了X = 7.72 μ m、Y = 5.79 μ m和Z = 3.81 μ m的体积，横向像素尺寸为7.5 nm，Z切片厚度为30 nm。膜的内在对比度足以区分线粒体内的高尔基体池、囊泡、内质网和嵴等结构，并允许在少突胶质细胞和星形胶质细胞过程中对不同类型的线粒体进行三维重建。把这项技术应用到休眠的B身上。我们获得了含有大量孢子的体积，并在核心中发现了明亮的信号，这与迄今为止任何已知的结构都不相关。总之，我们描述了使用冷冻FIB-SEM作为一种工具，用于直接和快速的3D冷冻成像的大型天然冷冻样品，包括组织。(C)2013 Elsevier Inc. All rights reserved.
Volume microscopy at high resolution is increasingly required to better understand cellular functions in the context of three-dimensional assemblies. Focused ion beam (FIB) milling for serial block face imaging in the scanning electron microscope (SEM) is an efficient and fast method to generate such volume data for 3D analysis. Here, we apply this technique at cryo-conditions to image fully hydrated frozen specimen of mouse optic nerves and Bacillus subtilis spores obtained by high-pressure freezing (HPF). We established imaging conditions to directly visualize the ultrastructure in the block face at -150 degrees C by using an in-lens secondary electron (SE) detector. By serial sectioning with a focused ion beam and block face imaging of the optic nerve we obtained a volume as large as X = 7.72 mu m, Y = 5.79 mu m and Z = 3.81 mu m with a lateral pixel size of 7.5 nm and a slice thickness of 30 nm in Z. The intrinsic contrast of membranes was sufficient to distinguish structures like Golgi cisternae, vesicles, endoplasmic reticulum and cristae within mitochondria and allowed for a three-dimensional reconstruction of different types of mitochondria within an oligodendrocyte and an astrocytic process. Applying this technique to dormant B. subtilis spores we obtained volumes containing numerous spores and discovered a bright signal in the core, which cannot be related to any known structure so far. In summary, we describe the use of cryo FIB-SEM as a tool for direct and fast 3D cryo-imaging of large native frozen samples including tissues. (C) 2013 Elsevier Inc. All rights reserved.