Chang Irene Y T, Joester Derk
Department of Materials Science and Engineering, Northwestern University, 2220 Campus Drive, Evanston, IL 60208, USA.
J Struct Biol. 2015 Dec;192(3):569-579. doi: 10.1016/j.jsb.2015.11.002. Epub 2015 Nov 6.
Cryo-SEM is a high throughput technique for imaging biological ultrastructure in its most pristine state, i.e. without chemical fixation, embedding, or drying. Freeze fracture is routinely used to prepare internal surfaces for cryo-SEM imaging. However, the propagation of the fracture plane is highly dependent on sample properties, and the resulting surface frequently shows substantial topography, which can complicate image analysis and interpretation. We have developed a broad ion beam milling technique, called cryogenic triple ion gun milling (CryoTIGM™ ['krī-ə-,tīm]), for cryo-planing frozen-hydrated biological specimens. Comparing sample preparation by CryoTIGM™ and freeze fracture in three model systems, Baker's yeast, mouse liver tissue, and whole sea urchin embryos, we find that CryoTIGM™ yields very large (∼700,000 μm(2)) and smooth sections that present ultrastructural details at similar or better quality than freeze-fractured samples. A particular strength of CryoTIGM™ is the ability to section samples with hard-soft contrast such as brittle calcite (CaCO3) spicules in the sea urchin embryo.
低温扫描电子显微镜（Cryo-SEM）是一种高通量技术，用于对处于最原始状态的生物超微结构进行成像，即无需化学固定、包埋或干燥。冷冻断裂通常用于为低温扫描电子显微镜成像制备内表面。然而，断裂平面的扩展高度依赖于样品特性，并且所得到的表面常常呈现出显著的形貌，这可能会使图像分析和解释变得复杂。我们开发了一种宽离子束铣削技术，称为低温三离子枪铣削（CryoTIGM™ ['krī-ə-,tīm]），用于对冷冻水合生物标本进行低温刨削。在三个模型系统（面包酵母、小鼠肝脏组织和整个海胆胚胎）中比较了通过CryoTIGM™和冷冻断裂进行的样品制备，我们发现CryoTIGM™能产生非常大（约700,000μm²）且光滑的切片，其呈现的超微结构细节质量与冷冻断裂样品相似或更好。CryoTIGM™的一个特别优势是能够对具有硬-软对比度的样品进行切片，例如海胆胚胎中的脆性方解石（CaCO₃）针状物。
