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

冷冻电镜中冷冻水合标本的冷冻超显微切片术

• 批准号: 7722822
• 负责人: MARK S LADINSKY
• 金额: $ 3.69万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7722822
• 关键词: Address; Biological Preservation; Cell Wall; Cells; Computer Retrieval of Information on Scientific Projects Database; Cryoelectron Microscopy; Cryoultramicrotomy; Custom; Decompression Sickness; Diamond; Electron Microscope; Equilibrium; Face; Fracture; Freezing; Funding; Goals; Grant; Institution; Mammalian Cell; Morphologic artifacts; Reproducibility; Research; Research Personnel; Resources; Sampling; Shapes; Source; Specimen; Standards of Weights and Measures; Surface; Temperature; Thick; Tissues; Trapezoid bone structure; United States National Institutes of Health; Work; Yeasts; beta-Glucans; brass; improved; pressure; size; tomography; tool

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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 我们正在努力提高水合冷冻切除术的质量和可重复性，从高压冷冻（HPF）组织或细胞的块切割，以制备用于冷冻光学显微镜的标本。 制备样品块，以便它们保持在两件式黄铜HPF帽子的平坦半部。 然后将帽子安装在黄铜夹子中，该夹子适合Leica Ultracut-uct/fcs Croymicrotome的标本Chuck。 将块修剪并面对定制的Diatome钻石修剪工具，并用硅藻级别的Cryo-P钻石刀切割。 我们最好的刀在刀边缘上有25o的角度。 钻石刀本身是两种类型的人造伪影，共有水合冷冻切除术。截面与切割方向和截面的断裂相平行的压缩，因为截面从刀边缘弯曲时。 当电子显微镜中查看时，这些裂缝在截面表面中显示为“波纹”。 该项目的主要目标是减少部分的表面断裂。尽管我们尚未消除这种伪像，但通过解决刀角以外的几个因素，我们大大降低了它。 其中包括减小样品块面的大小，将面部呈垂直矩形而不是水平梯形，调整截面厚度并密切控制截面室内的静态。 此外，我们发现酵母菌的颗粒比哺乳动物细胞的颗粒更高的质量和可重复性。当哺乳动物细胞与HPF之前的酵母量相等的酵母混合时，所产生的冷冻切除率与纯酵母样品相似。对酵母细胞的玻璃体切片的仔细检查表明，细胞壁（尤其是内部最大层）显示表面裂缝显着减少，或者根本没有断裂。因此，我们正在探索用作主要细胞壁成分的分子，例如β-葡聚糖，用作冻结培养基代替标准冷冻保护剂。 我们还将研究可以在分段质量/可重现性和样品保存之间提供最佳平衡的分段室温度的程度。