USE OF FIB FOR PREPARATION OF FROZEN-HYDRATED SPECIMENS

使用 FIB 制备冷冻水合样本

• 批准号: 8172270
• 负责人: MICHAEL MARKO
• 金额: $ 5.41万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-07 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8172270
• 关键词: Agreement; Bacteria; Biocompatible Materials; Biological; Caliber; Cells; Collaborations; Computer Retrieval of Information on Scientific Projects Database; Cryoelectron Microscopy; Custom; Cyanobacterium; Development; Disclosure; Electrons; Engineering; Equipment; Escherichia coli; Freezing; Funding; Goals; Grant; Ice; Image; Institution; Ions; Liquid substance; Mechanics; Methods; Modification; Morphologic artifacts; Nanotechnology; Nature; Nitrogen; Paper; Phase; Preparation; Procedures; Reporting; Research; Research Personnel; Resources; Salmon; Sampling; Scientist; Small Business Innovation Research Grant; Source; Specimen; Staging; Staging System; Structure; Surface; Suspension substance; Suspensions; System; Techniques; Temperature; Testing; Thick; Time; Tissue Sample; Tissues; Tomogram; Travel; Ultramicrotomy; United States National Institutes of Health; Universities; Water; Work; Writing; abstracting; base; college; design; electron tomography; instrument; novel; pressure; prototype; research study; tomography

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. ABSTRACT: Because of inherent difficulties associated with cryo-ultramicrotomy, we have begun a highly novel, parallel approach to generate thin specimens of bulk biological tissue and whole cells: the use of a focused ion beam (FIB) to mill the specimens. The first experiments, carried at the Harvard Center for Imaging of Mesoscale Structures, confirmed that vitreously-frozen water can be milled with the ion beam without devitrification: + Marko, M., Hsieh, C., MoberlyChan, W., Mannella, C., Frank, J. (2006) Focused ion beam milling of vitreous water: prospects for an alternative to cryo-ultramicrotomy. J. Microsc. 222(1) 42-47. This work represents a parallel line of development that avoids many of the technical difficulties associated with mechanical sectioning, including section compression, surface artifacts, and attachment problems. In order to demonstrate that biological material can be FIB-milled for subsequent cryo-electron tomography, we used plunge-frozen suspensions of bacteria on TEM grids. The bacteria (E. coli and cyanobacteria) were between 500 and 1000 nm in diameter, and the ice layer was in excess of 1000 nm in thickness. We cut the TEM grids in half under liquid nitrogen, and FIB-milled normal to the cut edge, thinning the frozen suspension to 200-500 nm. We performed electron tomography on several samples that were thinned to 500 nm in thickness. The specimens remained vitreous (based on electron diffraction), and the tomograms revealed no obvious signs of damage at the cut surface. + Marko, M., Hsieh, C., Schalek, R., Frank, J. and Mannella, C.A. (2007). Focused-ion-beam thinning of frozen-hydrated biological specimens for cryo-electron microscopy. Nature Methods: 4(3): 215-217. The major current effort in the FIB project is to develop convenient procedures for milling high-pressure frozen tissue for TEM tomography. We are collaborating with Hummingbird Scientific in this effort. This company, led by mechanical engineer Norman Salmon and materials scientist Eric Stach of Purdue, specializes in TEM and SEM specimen holders and stages. Hummingbird has written several successful NIH Phase I and Phase II SBIR proposals with our collaboration. Non-disclosure and cooperative research agreements are in effect for this development work. A prototype FIB cryo-stage system was purchased by the Resource in December 2006, and has been installed at the University of Albany's College of Nanoscale Science and Engineering on an FEI Nanolab V600 FIB/SEM instrument. The system includes special fixtures and equipment to take tissue samples from the high-pressure freezer, through the FIB, and into the TEM, while keeping the specimen below the devitrification temperature and free of frost at all times. During the last reporting period, M. Marko traveled extensively, giving invited talks on this new technique in biological cryo-EM. We have tested all the components of the Hummingbird cryo-FIB system, and suggested several modifications that were subsequently implemented. Our goal for this reporting period was to apply the technique to high-pressure frozen pelleted cells or blocks of tissue. We froze mammalian (RBC) cell pellets in custom high-pressure freezer carriers designed to fit in a TEM cryo-transfer specimen holder. To demonstrate that specimens can be vitreously frozen in the custom holders, we cut excellent frozen-hydrated cryo-ultramicrotome sections. As reported in our most recent paper on cryo-ultramicrotomy (Hsieh et al, 2006, J. Struct. Biol. 153:1-13), only the highest quality freezing can yield excellent frozen-hydrated sections.

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。 摘要： 由于冷冻超微切割固有的困难，我们已经开始了一种非常新颖的、平行的方法来产生大量生物组织和整个细胞的薄标本：使用聚焦离子束(FIB)来研磨样品。在哈佛大学中尺度结构成像中心进行的第一批实验证实，玻璃体冻结的水可以用离子束研磨而不会析出玻璃化： +Marko，M.，Hsieh，C.，MoberlyChan，W.，Mannella，C.，Frank，J.(2006)玻璃体水的聚焦离子束研磨：冷冻超微切割术的替代方案的前景。J.Microsc.222(1)42-47 这项工作代表了一条平行的发展路线，它避免了许多与机械剖切相关的技术困难，包括截面压缩、表面伪影和附着问题。 为了证明生物材料可以被FIB碾磨用于随后的冷冻电子断层扫描，我们使用了在电子显微镜网格上插入冷冻的细菌悬浮液。细菌(大肠杆菌和蓝藻)直径在500~1000 nm之间，冰层厚度超过1000 nm。我们在液氮下将电子显微镜网格切成两半，并垂直于切割边缘进行FIB球磨，将冷冻悬浮液稀释至200-500 nm。我们对几个厚度变薄到500纳米的样品进行了电子断层扫描。标本仍然是玻璃体(基于电子衍射)，断层图像显示切割表面没有明显的损伤迹象。 +Marko，M.，Hsieh，C.，Schalek，R.，Frank，J.和Mannella，C.A.(2007)。冷冻电子显微镜用冷冻水合生物样品的聚焦离子束减薄。自然方法：4(3)：215-217。 目前FIB项目的主要工作是开发用于瞬变断层扫描的高压冷冻组织的简便加工程序。我们正在与蜂鸟科学公司在这方面进行合作。这家公司由机械工程师诺曼·萨蒙和普渡大学的材料科学家埃里克·斯塔奇领导，专门从事透射电子显微镜和扫描电子显微镜样品架和工作台的研究。与我们的合作，蜂鸟已经成功地撰写了几份NIH第一阶段和第二阶段SBIR提案。保密协议和合作研究协议对这项开发工作有效。该资源公司于2006年12月购买了FIB低温工作台原型系统，并已安装在奥尔巴尼大学纳米科学与工程学院的FEI NanoLab V600 FIB/SEM仪器上。该系统包括特殊的固定装置和设备，用于从高压冷冻箱中采集组织样本，通过FIB，并进入透射电子显微镜，同时将样本保持在非玻璃化温度以下，并且始终没有霜冻。在上一次报告所述期间，M.Marko进行了广泛的旅行，就这项生物低温EM新技术作了应邀演讲。 我们已经测试了蜂鸟冷冻-FIB系统的所有部件，并提出了几项修改建议，随后实施了这些修改。我们在本报告期间的目标是将这项技术应用于高压冷冻的丸状细胞或组织块。我们将哺乳动物(RBC)细胞颗粒冷冻在定制的高压冷冻载体中，该载体专为适合TEM低温转移标本支架而设计。为了证明标本可以在定制保持器中玻璃化冷冻，我们切下了极好的冷冻水合冷冻超微切片。正如我们最近发表的关于冷冻-超显微手术的论文(Hsieh et al，2006，J.Struct.比奥尔。153：1-13)，只有最高质量的冷冻才能产生优质的冷冻水化切片。