HVEM OPERATION AND IMPROVEMENTS

该子项目是利用 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来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 摘要： 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（另一个在日本冈崎）。 HVEM的最大加速电压为1200 kV，点对点分辨率约为5 º。 它配备了一些专门的样品架，内部建造，适用于单和双倾斜断层扫描高达75 <$倾斜。 也可以使用侧入式差压泵环境室。 内部设计的增强视频速率CCD电视系统可用于观察具有非常低的电子辐射的图像，用于在断层扫描数据收集期间聚焦和跟踪图像。 设想了几个改进领域以增加HVEM的有用性。 其中包括倾斜台的计算机控制和冷冻断层扫描的实施。 用于电视的摄像机足够灵敏，可以在高达10，000倍的低温断层扫描期间手动聚焦和跟踪，并且现有的冷冻转移阶段可能会被修复。 对速冻全细胞进行HVEM冷冻断层扫描将补充对此类细胞外周的IVEM进行的工作。 一个优秀的，专门设计的CCD相机是从博尔德NCRR资源后，他们的HVEM退役。 在我们的HVEM上安装此摄像机将简化断层倾斜系列的收集。 HVEM用于厚切片的电子断层扫描有两个重要的优点，但通常不被重视。 首先，HVEM具有大的焦距，因此具有大的焦深。 因此，5000 nm厚的染色塑料切片可以倾斜60度，而不会在图像边缘产生模糊，而在倾斜的250 nm厚的染色塑料切片的IVEM图像中可以看到一些模糊。 其次，HVEM的厚染色塑料切片的电子剂量通常比IVEM低三倍，因为IVEM需要非常小的物镜孔径来实现倾斜厚切片的足够焦深。

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.