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: 7597300
• 负责人: MARK S LADINSKY
• 金额: $ 2.3万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7597300
• 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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我们正在努力提高从高压冷冻（HPF）组织或细胞块切割的水合冷冻切片的质量和再现性，以便为冷冻电子显微镜制备标本。 准备样品块，使它们保持在两件式黄铜HPF帽的平坦半部分中的圆顶。 然后将帽子安装在一个黄铜夹头中，该夹头与Leica UltraCut-UCT/FCS冷冻切片机的样本卡盘相匹配。 该块是修剪和面对一个定制的Diatome钻石修剪工具，和部分是用Diatome Cryo-P钻石刀切割。 我们最好的刀在刀刃处有25度的夹角。 金刚石刀本身是水合冷冻切片常见的两种类型伪影的重要贡献者;平行于切割方向的切片压缩和远离刀刃弯曲时的切片断裂。 这些骨折看起来 涟漪 当在电子显微镜下观察时， 该项目的一个主要目标是减少断面的表面破裂。虽然我们还没有消除这种伪影，但我们通过解决刀角以外的几个因素，已经大大减少了这种伪影。 这些措施包括减小样品块表面的尺寸，将表面成形为垂直矩形而不是水平梯形，调整切片厚度和严格控制切片室内的静电。 此外，我们还发现，酵母细胞团产生的切片质量和重现性比哺乳动物细胞团高得多;当哺乳动物细胞在HPF之前与等体积的酵母混合时，所得冷冻切片的质量与纯酵母样品相似。对酵母细胞的玻璃体切片的仔细检查显示，细胞壁（特别是最内层）显示出表面破裂的显著减少，或者根本没有破裂。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，作为冷冻介质，代替标准的冷冻保护剂。 我们还将研究切片室的温度可以变化的程度，以提供切片质量/再现性和样品保存之间的最佳平衡。