Practical workflow for cryo focused-ion-beam milling of tissues and cells for cryo-TEM tomography.
Practical workflow for cryo focused-ion-beam milling of tissues and cells for cryo-TEM tomography.
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DOI：
10.1016/j.jsb.2013.10.019
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发表时间：
2014-01
影响因子：
3
通讯作者：
Marko, Michael
中科院分区：
文献类型：
--
作者：
Hsieh, Chyongere;Schmelzer, Thomas;Kishchenko, Gregory;Wagenknecht, Terence;Marko, Michael
玻璃体冷冻提供了一种通过冷冻透射电子显微镜（cryo-TEM）研究接近天然状态的细胞和组织的方法，这在需要大分子水平的结构信息时非常重要。许多细胞-特别是组织中的细胞-太厚，无法在冷冻TEM中完整地研究。低温聚焦离子束（cryo-FIB）铣削在一些实验室中用于薄化玻璃体冷冻标本，从而避免了冷冻超薄切片的伪影和困难。然而，该技术是具有挑战性的，因为需要在整个过程中避免失透和结霜，从最初的冷冻步骤到将样品加载到低温TEM的最后步骤。我们提出了一个强大的工作流程，利用定制的夹具和设备，可用于高压冷冻散装组织样品以及TEM网格上冷冻的样品。
Vitreous freezing offers a way to study cells and tissue in a near-native state by cryo-transmission electron microscopy (cryo-TEM), which is important when structural information at the macromolecular level is required. Many cells -- especially those in tissue -- are too thick to study intact in the cryo-TEM. Cryo focused-ion-beam (cryo-FIB) milling is being used in a few laboratories to thin vitreously frozen specimens, thus avoiding the artifacts and difficulties of cryo-ultramicrotomy. However, the technique is challenging because of the need to avoid devitrification and frost accumulation during the entire process, from the initial step of freezing to the final step of loading the specimen into the cryo-TEM. We present a robust workflow that makes use of custom fixtures and devices that can be used for high-pressure-frozen bulk tissue samples as well as for samples frozen on TEM grids.
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