In-situ integrity control of frozen-hydrated, vitreous lamellas prepared by the cryo-focused ion beam-scanning electron microscope
In-situ integrity control of frozen-hydrated, vitreous lamellas prepared by the cryo-focused ion beam-scanning electron microscope
复制标题
DOI：
10.1016/j.jsb.2013.05.016
复制
发表时间：
2013-07-01
影响因子：
3
通讯作者：
Post, Jan A.
中科院分区：
文献类型：
--
作者：
de Winter, D. A. Matthijs;Mesman, Rob J.;Post, Jan A.
最近，已经提出了一些新的方法，其意图是使用适用于低温透射电子显微镜（cryo-TEM）的聚焦离子束（FIB）系统从水合的玻璃质冷冻的低温样品产生电子束透明的低温切片（FIB-SEM术语中的薄片）。由于工作流程仍然具有挑战性且耗时，因此能够确定薄片的完整性和适用性（细胞与无细胞;玻璃体与结晶）非常重要。在这里，我们提出了一种原位方法，测试这两个条件下使用低温扫描电子显微镜（低温扫描电子显微镜）在透射模式（TSEM;透射扫描电子显微镜），一旦FIB制成的薄片准备好。未染色细胞的Cryo-TSEM成像产生强烈的对比度，使得能够直接成像存在于板层中的材料。此外，取向对比度被证明是适合于区分结晶片层从玻璃体片层。将载物台倾斜几度导致冰粒之间的对比度作为倾斜角的函数发生变化，而具有玻璃状冰的区域的对比度作为倾斜角的函数保持不变。这种取向对比度随后通过透射模式下的低温电子背散射衍射（EBSD）进行了验证。集成所提出的方法进行了讨论，它可以在未来的发展中发挥的作用，一个新的和创新的所有功能于一体的低温FIB-SEM生命科学仪器。(C)2013 Elsevier Inc. All rights reserved.
Recently a number of new approaches have been presented with the intention to produce electron beam transparent cryo-sections (lamellas in FIB-SEM terminology) from hydrated vitreously frozen cryo samples with a Focused Ion Beam (FIB) system, suitable for cryo-Transmission Electron Microscopy (cryo-TEM). As the workflow is still challenging and time consuming, it is important to be able to determine the integrity and suitability (cells vs. no cells; vitreous vs. crystalline) of the lamellas. Here we present an in situ method that tests both conditions by using the cryo-Scanning Electron Microscope (cryo-SEM) in transmission mode (TSEM; Transmission Scanning Electron Microscope) once the FIB-made lamella is ready. Cryo-TSEM imaging of unstained cells yields strong contrast, enabling direct imaging of material present in the lamellas. In addition, orientation contrast is shown to be suitable for distinguishing crystalline lamellas from vitreous lamellas. Tilting the stage a few degrees results in changes of contrast between ice grains as a function of the tilt angle, whereas the contrast of areas with vitreous ice remains unchanged as a function of the tilt angle. This orientation contrast has subsequently been validated by cryo-Electron BackScattered Diffraction (EBSD) in transmission mode. Integration of the presented method is discussed and the role it can play in future developments for a new and innovative all-in-one cryo-FIB-SEM life sciences instrument. (C) 2013 Elsevier Inc. All rights reserved.