HVEM OPERATION AND IMPROVEMENTS

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： 奥尔巴尼Hvem是美洲最后一家，也是世界上仅有的两家致力于生物学研究的机构之一(另一家位于日本冈崎)。HVEM的最大加速电压为1200KV，点对点分辨率约为5？它配备了许多专门的样品夹持器，由内部制造，适用于高达75°的单倾斜和双倾斜层析成像。还提供侧向进气式差动泵浦环境室。采用自行设计的增强型视频率CCD电视系统，可以在极低的电子辐照下观察图像，在层析数据采集过程中对图像进行聚焦和跟踪。 设想了几个改进领域，以提高清洁发展机制的有用性。其中包括倾斜阶段的计算机控制和低温层析成像的实施。用于电视观看的摄像机的灵敏度足以在冷冻断层扫描过程中进行手动对焦和跟踪，最高可达10,000倍，现有的冷冻传输阶段很可能可以修复。对急速冷冻的整个细胞进行的高压电子显微镜冷冻断层扫描将补充在这类细胞外围的IVEM上所做的工作。 在Boulder NCRR资源的HVEM退役后，获得了一台出色的、专门设计的CCD相机。这款相机安装在我们的HVEM上，将简化断层扫描倾斜系列的收集。 对于厚切片的电子层析成像，HVEM有两个重要的优点，这两个优点往往没有得到重视。首先，HVEM具有较大的焦距，因此具有较大的焦深。因此，厚达5000 nm的染色塑料切片可以倾斜到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图像中会看到一些模糊。其次，彩色塑料厚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才能对倾斜的厚片实现足够的焦深。

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.