USE OF FIB FOR PREPARATION OF FROZEN-HYDRATED SPECIMENS

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。子项目及 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，我们正在从事一个非常新颖的，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。 第一个实验是由哈佛中尺度结构成像中心的Warren MoberlyChan博士进行的，实验证实了玻璃状冷冻水可以用离子束研磨而不会失透。 + Marko，M.，Hsieh，C.，MoberlyChan，W.，曼内拉角，和Frank，J.（2006年）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。 这代表了一条平行的发展路线，原则上，将避免与机械切片相关的许多技术困难，包括切片压缩、表面伪影和附着问题。 制定了制备标本的一般策略，涉及解决几个技术问题。 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），其中直径在500和1000 nm之间，并且冰层厚度超过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.，Hsieh，C.，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项目的一个主要工作是制定出方便地研磨高压冷冻组织进行TEM断层扫描的程序。 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与我们合作者一起编写了成功的NIH第一阶段和第二阶段SBIR提案，我们已经签署了这项开发工作的保密文件。 2006年12月，资源公司购买了一个原型系统，并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。 该系统的设计包括特殊的夹具和设备，用于从高压冷冻机中取出组织样本，通过FIB，并进入TEM，同时保持样本低于失透温度，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。

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 the inherent difficulties associated with cryo-ultramicrotomy, we are engaging in 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 out with Dr. Warren MoberlyChan 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., and Frank, J. (2006) Focused ion beam milling of vitreous water: prospects for an alternative to cryo-ultramicrotomy. J. Microsc. 222(1)42-47. This represents a parallel line of development that, in principle, would avoid many of the technical difficulties associated with mechanical sectioning, including section compression, surface artifacts, and attachment problems. A general strategy for preparing the specimens was worked out, involving resolution of several technical 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) where 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. A major effort in the FIB project is to work out procedures for conveniently 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 a successful NIH Phase I and Phase II SBIR proposals, with us collaborators, and we have signed an non-disclosure document for this development work. A prototype 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 was designed to include 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.