低温聚焦离子束(FIB)铣削是一种强大的技术,细胞标本通过电子冷冻成像(cryo-ET)进行高分辨率结构分析。FIB研磨的薄片可以从网格上的细胞产生,或者从较厚的地方切下来,高压冷冻标本。然而,这些方法可以对样本施加几何约束,这可能是无用的,特别是当成像细胞内具有非常明确的取向的结构时。例如,插入冷冻杆状细菌平行于网格平面,然而Z形环,微管蛋白样蛋白FtsZ的丝状结构和细菌分裂的关键组织者,围绕细胞的圆周运行,使其垂直于成像平面。因此,用当前技术对许多完整的环成像是困难或不切实际的。为了避免这个问题,我们已经用蜂窝状几何形状的规则排列的圆柱形孔制造了整体金标本载体,以垂直方向捕获细菌。这些支持,我们称之为“蜂窝状金盘”,替换标准EM网格,当与FIB铣削结合时,可以生产包含穿过细胞的横截面的薄片。所得薄片比常规薄片更稳定且耐破损和带电。蜂窝状圆盘的设计可以根据需要进行修改,因此也将使其他样本的低温-ET和低温-EM成像在其他情况下难以获得的方向。
Cryo-focussed ion beam (FIB)-milling is a powerful technique that opens up thick, cellular specimens to high-resolution structural analysis by electron cryotomography (cryo-ET). FIB-milled lamellae can be produced from cells on grids, or cut from thicker, high-pressure frozen specimens. However, these approaches can put geometrical constraints on the specimen that may be unhelpful, particularly when imaging structures within the cell that have a very defined orientation. For example, plunge frozen rod-shaped bacteria orient parallel to the plane of the grid, yet the Z-ring, a filamentous structure of the tubulin-like protein FtsZ and the key organiser of bacterial division, runs around the circumference of the cell such that it is perpendicular to the imaging plane. It is therefore difficult or impractical to image many complete rings with current technologies. To circumvent this problem, we have fabricated monolithic gold specimen supports with a regular array of cylindrical wells in a honeycomb geometry, which trap bacteria in a vertical orientation. These supports, which we call \"honeycomb gold discs\", replace standard EM grids and when combined with FIB-milling enable the production of lamellae containing cross-sections through cells. The resulting lamellae are more stable and resistant to breakage and charging than conventional lamellae. The design of the honeycomb discs can be modified according to need and so will also enable cryo-ET and cryo-EM imaging of other specimens in otherwise difficult to obtain orientations.