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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们正在努力提高从高压冷冻(HPF)组织或细胞块中切下的水合冰冻切片的质量和重复性，以便为冷冻电子显微镜准备标本。样品块被准备好，以便它们保持在两件式黄铜HPF帽子的平坦的一半中。然后将帽子安装在黄铜夹头中，该夹头适合徕卡UltraCut-UCT/FCS冷冻微型机的标本卡盘。该块是修剪和面对一个定制的Diatome钻石修剪工具，部分是用Diatome Cryo-P钻石刀切割。我们最好的刀刃有一个25度的夹角。金刚石刀本身是水合冷冻切割常见的两种伪影的重要因素：平行于切割方向的部分被压缩，以及当部分弯曲离开刀刃时断裂。当在电子显微镜下观察时，这些断口在截面表面显示为“波纹”。该项目的一个主要目标是减少节段的表面破裂。虽然我们还没有消除这一伪影，但我们已经通过解决除刀具角度之外的其他几个因素显著减少了它。这些措施包括缩小试块表面的尺寸，将表面成形为垂直矩形而不是水平梯形，调整截面厚度和密切控制切割室内的静电。此外，我们还发现，酵母颗粒比哺乳动物细胞颗粒产生的切片质量和重复性要高得多；当哺乳动物细胞在HPF之前与同等体积的酵母混合时，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观察发现，细胞壁(特别是最内层)的表面断裂显著减少，或根本没有断裂。因此，我们正在探索使用作为主要细胞壁成分的分子，如β-葡聚糖，取代标准的冷冻保护剂作为冷冻介质。我们还将调查切片室的温度可以变化的程度，以便在切片质量/重复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.