HVEM OPERATION AND IMPROVEMENTS

HVEM 操作和改进

• 批准号: 7721704
• 负责人: DAVID BARNARD
• 金额: $ 5.56万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7721704
• 关键词: Americas; Area; Biological; Blur; Cells; Collection; Complement; Computer Retrieval of Information on Scientific Projects Database; Computers; Data Collection; Depth; Dose; Electrons; Freezing; Funding; Grant; Housing; Image; Institution; Japan; Length; Manuals; National Center for Research Resources; Numbers; Operative Surgical Procedures; Plastics; Pump; Rate; Research; Research Personnel; Resolution; Resources; Series; Side; Source; Specimen; Staging; Staining method; Stains; System; Thick; Time; United States National Institutes of Health; Work; abstracting; charge coupled device camera; design; electron tomography; high voltage electron microscopy; irradiation; repaired; tomography; voltage

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: The Albany HVEM is the last in the Americas, and one of only two in the world that are dedicated to biological studies (the other is in Okazaki, Japan). The HVEM has a maximum accelerating voltage of 1200kV, and a point-to-point resolution of about 5¿. It is equipped with a number of specialized specimen holders, built in-house, that are suitable for single and double-tilt tomography up to 75¿ tilt. A side-entry differentially-pumped environmental chamber is also available. An in-house designed intensified video-rate CCD TV system can be used to observe the image with very low electron irradiation, for focusing and following the image during tomographic data collection. Several areas of improvement are envisioned for increasing the usefulness of the HVEM. Among these are computer control of the tilt stage and implementation of cryo-tomography. The video camera used for televiewing is sensitive enough for manual focus and tracking during cryo-tomography at up to about 10,000X, and the existing cryotransfer stage can likely be repaired. HVEM cryo-tomography of plunge-frozen whole cells will complement work done on the IVEM on the periphery of such cells. An excellent, specially-designed CCD camera was obtained from the Boulder NCRR Resource after their HVEM was decommissioned. Installation of this camera on our HVEM will streamline collection of tomographic tilt series. There are two important benefits of the HVEM for electron tomography of thick sections that are often not appreciated. First, the HVEM has a large focal length and so a large depth of focus. Thus, stained plastic sections as thick as 5000 nm can be tilted to 60 degrees without producing blurring at the edges of the image, whereas some blurring is seen in IVEM images of tilted, 250-nm-thick stained plastic sections. Second, the electron dose with thick stained plastic sections is typically three times lower with the HVEM than with the IVEM, since the latter needs a very small objective aperture to achieve adequate depth of focus for tilted thick sections.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 列出的机构是。 对于中心来说，它不一定是研究者的机构。 抽象的： 奥尔巴尼 HVEM 是美洲最后一个，也是世界上仅有的两个致力于生物学研究的 HVEM 之一（另一个位于日本冈崎）。HVEM 的最大加速电压为 1200kV，点对点。点分辨率约为 5¿它配备了许多内部内置的专用样品架，适用于高达 75° 的单倾斜和双倾斜断层扫描还可以使用侧入差动泵浦环境室，使用内部设计的增强视频速率 CCD 电视系统以非常低的电子辐射观察图像，以便在断层扫描数据收集期间聚焦和跟踪图像。 。 为了提高 HVEM 的实用性，我们设想了几个改进领域，其中包括倾斜台的计算机控制和冷冻断层扫描的实施。用于电视观看的摄像机对于在冷冻断层扫描期间进行手动对焦和跟踪足够敏感。大约 10,000 倍，并且现有的冷冻转移阶段可能会得到修复，对冷冻全细胞的 HVEM 冷冻断层扫描将补充在此类外围的 IVEM 上所做的工作。细胞。 在博尔德 NCRR 资源退役后，我们获得了一台出色的、专门设计的 CCD 相机。在我们的 HVEM 上安装该相机将简化断层摄影倾斜系列的收集。 HVEM 对于厚切片的电子断层扫描有两个通常不被重视的重要优点：首先，HVEM 具有较大的焦距，因此可以倾斜厚达 5000 nm 的染色塑料切片。到 60 度，图像边缘不会产生模糊，而在倾斜的 250 nm 厚染色塑料切片的 IVEM 图像中可以看到一些模糊。 其次，厚染色塑料的电子剂量。 HVEM 的截面通常比 IVEM 低三倍，因为后者需要非常小的物镜孔径才能为倾斜的厚截面实现足够的焦深。