HVEM OPERATION AND IMPROVEMENTS

这个子项目是众多研究子项目之一

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.