Molecular Structure and Function Program, The Hospital for Sick Children Research Institute, Toronto, Canada.
Molecular Structure and Function Program, The Hospital for Sick Children Research Institute, Toronto, Canada; Department of Biochemistry, The University of Toronto, Canada; Department of Medical Biophysics, The University of Toronto, Canada.
J Struct Biol. 2014 Jan;185(1):42-7. doi: 10.1016/j.jsb.2013.11.002. Epub 2013 Nov 21.
Single particle electron cryomicroscopy (cryo-EM) is often performed using EM grids coated with a perforated or holey layer of amorphous carbon. Regular arrays of holes enable efficient cryo-EM data collection and several methods for the production of micropatterned holey-carbon film coated grids have been described. However, a new generation of direct detector device (DDD) electron microscope cameras can benefit from hole diameters that are smaller than currently available. Here we extend a previously proposed method involving soft lithography with a poly(dimethylsiloxane) (PDMS) stamp for the production of holey-carbon film coated EM grids. By incorporating electron-beam (e-beam) lithography and modifying the procedure, we are able to produce low-cost high-quality holey-carbon film coated EM grids with ∼500nm holes spaced 4μm apart centre-to-centre. We demonstrate that these grids can be used for cryo-EM. Furthermore, we show that by applying image shifts to obtain movies of the carbon regions beside the holes after imaging the holes, the contrast transfer function (CTF) parameters needed for calculation of high-resolution cryo-EM maps with a DDD can be obtained efficiently.
单颗粒电子低温冷冻透射显微镜（cryo-EM）通常使用涂有穿孔或有孔无定形碳层的 EM 网格进行。规则的孔阵列能够实现高效的 cryo-EM 数据收集，并且已经描述了几种生产微孔碳膜涂覆网格的方法。然而，新一代直接探测器装置（DDD）电子显微镜相机可以受益于比目前可用的孔径更小的孔径。在这里，我们扩展了一种先前提出的方法，该方法涉及使用聚二甲基硅氧烷（PDMS）印章进行软光刻，以生产涂有碳膜的 EM 网格。通过结合电子束（e-beam）光刻和修改该过程，我们能够以低成本生产高质量的涂有碳膜的 EM 网格，其孔直径约为 500nm，中心到中心的间距为 4μm。我们证明了这些网格可用于 cryo-EM。此外，我们还表明，通过在对孔成像后应用图像移位来获得孔旁碳区域的电影，可以有效地获得用于 DDD 计算高分辨率 cryo-EM 图谱所需的对比传递函数（CTF）参数。
