CRYOULTRAMICROTOMY OF FROZEN-HYDRATED SPECIMENS IN CRYO-EM & TOMOGRAPHY

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。我们正在努力提高从高压冷冻（HPF）组织或细胞块中切割的水合冷冻切片的质量和可重复性，以便为冷冻电子显微镜准备标本。准备好样品块，使它们在两件式黄铜HPF帽的扁平一半中保持圆顶。然后将帽子安装在黄铜夹套中，该夹套适合徕卡UltraCut-UCT/FCS冷冻微生物组的标本夹头。该块是修剪和面对一个定制的硅藻金刚石修剪工具，部分是切割硅藻低温- p金刚石刀。我们最好的刀刀刃的夹角是250度。钻石刀本身是水合冷冻切片常见的两种人工制品的重要贡献者；平行于切割方向的截面的压缩和当它从刀口弯曲时截面的断裂。当在电子显微镜下观察时，这些裂缝在断面表面表现为“波纹”。该项目的一个主要目标是减少井段的表面裂缝。虽然我们还没有消除这个伪影，但我们已经通过解决除刀角以外的几个因素显著地减少了它。这些措施包括减小试样块面尺寸，将面塑造成垂直矩形而不是水平梯形，调整切片厚度以及在切片室内严密控制静态。此外，我们发现酵母颗粒产量切片的质量和可重复性远高于哺乳动物细胞颗粒；当哺乳动物细胞与等量的酵母混合在HPF之前，所得到的冷冻切片在质量上与纯酵母样品相似。对酵母细胞玻璃体切片的仔细检查显示，细胞壁（尤其是最内层）表面裂缝明显减少，或者根本没有裂缝。因此，我们正在探索使用作为细胞壁主要成分的分子，如β -葡聚糖，作为代替标准冷冻保护剂的冷冻介质。我们还将研究切片室温度的变化程度，以便在切片质量/再现性和样品保存之间提供最佳平衡。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We are working to improve the quality and reproducibility of hydrated cryosections, cut from blocks of high-pressure frozen (HPF) tissues or cells, so as to prepare specimens for cryoelectron microscopy. Blocks of samples are prepared so they remain domed in the flat half of a two-piece brass HPF hat. The hat is then mounted in a brass collet that fits into the specimen chuck of a Leica UltraCut-UCT/FCS cryomicrotome. The block is trimmed and faced with a custom Diatome diamond trimming tool, and sections are cut with a Diatome Cryo-P diamond knife. Our best knife has a 25o included angle at the knife-edge. The diamond knife itself is a significant contributor to two types of artifact common to hydrated cryosections; compression of the section parallel to the cutting direction and fracturing of the section as it bends away from the knife edge. These fractures appear as ¿ripples¿ in the section surface when it is viewed in the electron microscope. A major goal of this project is to reduce the surface fracturing of sections. Although we have not eliminated this artifact, we have reduced it significantly by addressing several factors other than the knife angle. These include reducing the size of the specimen block face, shaping the face as a vertical rectangle rather than as a horizontal trapezoid, adjustment of section thickness and close control of static within the sectioning chamber. Furthermore, we have found that pellets of yeast yield sections of much higher quality and reproducibility than pellets of mammalian cells; when mammalian cells are mixed with an equal volume of yeast prior to HPF, the resulting cryosections are similar in quality to the pure yeast samples. Close inspection of vitreous sections of yeast cells revealed that the cell wall (especially the inner most layer) showed a significant reduction in the surface fractures, or no fracturing at all. We are therefore exploring the use of molecules that serve as major cell wall components, such as beta-glucans, as freezing media in place of standard cryoprotectants. We will also investigate the extent to which the temperature of the sectioning chamber can be varied in order to provide an optimal balance between section quality/reproducibility and sample preservation.