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。 摘要： 由于与冷冻超微切片术相关的固有困难，我们已经开始了一个非常新颖的，平行的方法来产生散装生物组织和全细胞的薄标本：使用聚焦离子束（FIB）磨标本。 在哈佛中尺度结构成像中心进行的第一个实验证实，玻璃冻结的水可以用离子束研磨而不会失透： + Marko，M.，Hsieh，C.，MoberlyChan，W.，曼内拉角，Frank，J.（2006）玻璃体水的聚焦离子束研磨：冷冻超微切片术替代方案的前景。J. Microsc. 222（1）42-47。 这项工作代表了一个平行的发展路线，避免了许多与机械切片相关的技术困难，包括部分压缩，表面工件和附件问题。 为了证明生物材料可以被FIB研磨以用于随后的低温电子断层扫描，我们在TEM网格上使用了插入冷冻的细菌悬浮液。 细菌（E.大肠杆菌和蓝细菌）的直径在500和1000 nm之间，冰层的厚度超过1000 nm。 我们在液氮下将TEM网格切成两半，并垂直于切割边缘进行FIB研磨，将冷冻悬浮液稀释至200-500 nm。 我们对几个样品进行了电子断层扫描，这些样品被减薄到500 nm的厚度。 样本保持玻璃状（基于电子衍射），断层图像显示切割表面无明显损伤迹象。 + Marko，M.，谢国忠，沙莱克河，Frank，J.和Mannella，C.A.（2007年）。冷冻电子显微镜用冷冻水合生物样品的聚焦离子束细化。 Nature Methods：4（3）：215-217. FIB项目目前的主要工作是开发用于TEM断层扫描的高压冷冻组织研磨的方便程序。 我们正在与Hummingbird Scientific合作开展这项工作。 该公司由机械工程师Norman Salmon和普渡大学的材料科学家Eric Stach领导，专门从事TEM和SEM样品架和载物台。 Hummingbird在我们的合作下已经成功地撰写了几份NIH第一阶段和第二阶段SBIR提案。 保密和合作研究协议对这项开发工作有效。 2006年12月，资源公司购买了一个原型FIB低温台系统，并将其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。 该系统包括特殊的夹具和设备，用于从高压冷冻机中取出组织样品，通过FIB，并进入TEM，同时保持样品低于失透温度，并始终无霜。 于上一报告期内，M. Marko广泛旅行，就生物冷冻EM中的这种新技术进行邀请演讲。 我们已经测试了蜂鸟冷冻FIB系统的所有组件，并提出了随后实施的几项修改。 本报告期内，我们的目标是将该技术应用于高压冷冻颗粒细胞或组织块。 我们将哺乳动物（RBC）细胞团冷冻在定制的高压冷冻器载体中，该载体设计成适合TEM冷冻转移标本保持器。 为了证明标本可以在定制的夹具中进行玻璃化冷冻，我们切割了出色的冷冻水合冷冻超薄切片机切片。 如我们最近关于冷冻超微切片术的论文（Hsieh et al，2006，J.Struct.Biol.153：1-13）中所报道的，只有最高质量的冷冻才能产生优异的冷冻水合切片。