GATAN IMAGING ENERGY FILTER WITH CCD CAMERA FOR IVEM

细胞生物学的前沿越来越多地集中在阐明细胞形态和组织的控制、细胞细胞器的性质和功能以及复杂蛋白质机器的作用。这些项目领域还为结构生物学定义了一个新的前沿，其目标不是阐明单个分子的结构，而是分子和超分子复合体以及细胞器的结构。电磁方法，如单粒子重建和电磁层析成像(EMT)，非常适合于分析这种大型结构。EMT单独适用于大型独特结构，如染色体、中心体、细胞骨架、神经元组织等。EMT单独适用于大型独特结构，如染色体、中心体、细胞骨架、神经元组织等。虽然加州大学旧金山分校在EMT方面取得了长足的进步，但我们的仪器不足以满足当前结构细胞生物学问题的要求。我们的目标是获得一种新的显微镜(菲利普斯300KVFEG He舞台)，配备能量过滤器和2Kx2K摄像机。作为这一昂贵过程的第一步，我们请求购买Gatan成像能量滤光片(GIF)和Gatan 2K x2K像素CCD相机。这种设备将立即在我们现有的显微镜上使用，并随时可以转移到新的显微镜上。收购能量过滤器和2Kx2K电荷耦合器件将创建一个收集高分辨率电磁断层扫描数据的设施，这在美国乃至世界上绝对是独一无二的。这一新设备将使我们能够1)对厚重的生物样品进行更高分辨率的层析成像，2)对更大的样品进行成像，3)使用CCD收集用于单粒子重建的高分辨率图像数据，4)从2D晶体收集高质量的衍射数据。这些好处源于使用GIF去除非弹性和多个非弹性散射电子，并且具有更大的面阵CCD，更适合200-300keV的成像。该设备将安装在我们现有的飞利浦300keV电子显微镜上，该电子显微镜位于加州大学旧金山分校生物化学系的电子显微镜实验室。该设施对整个园区以及外部用户都可用。除了两个之外，所有的核心用户目前都是由美国国立卫生研究院资助的，并且都在电子显微镜方面拥有丰富的经验。核心用户加上EM实验室的主管将组成一个咨询委员会，监督仪器的使用并制定政策。在核心调查员使用后的剩余时间内，将鼓励其他区域和国家用户使用。加州大学旧金山分校将为操作设备提供空间和人员支持(包括软件)。

Increasingly, the frontiers of cell biology focus on elucidating the control of cellular shape and organization, the nature and function of cellular organelles, and the role of complex protein machines. Such project areas also define a new frontier for structural biology where the goals are to elucidate not the structure of individual molecules, but structures of molecular and supramolecular complexes as well as organelles. EM methods such as single particle reconstruction and EM Tomography (EMT) are ideally suited to the analysis of such large structures. EMT alone is appropriate for large unique structures such as chromosomes, centrosomes, the cytoskeleton, neuronal organization, etc. EMT alone is appropriate for large unique structures such as chromosomes, centrosomes, the cytoskeleton, neuronal organization, etc. While great strides in the development of EMT have been made at UCSF, our instrumentation is inadequate to meet the demands of current problems in structural cell biology. Our goals are to acquire a new microscope (Phillips 300kV FEG He stage) equipped with an energy filter and a 2Kx2K CCD camera. As a first step in this expensive process we request the purchase of a Gatan Imaging Energy Filter (GIF) and a Gatan 2K x2K pixel CCD camera. This equipment will be immediately useful on our existing microscope and readily transferable to the new microscope. Acquisition of the energy filter and 2Kx2K CCD will create a facility for the collection of high resolution EM Tomography data that is absolutely unique in the US and perhaps the world. This new equipment will allow us to do 1) higher resolution tomography on thick biological samples, 2) image larger samples, 3) collect high resolution image data for single particle reconstruction using the CCD and 4) collect high quality diffraction data from 2D crystals. These benefits result from the use of the GIF to remove inelastic and multiple inelastic scattered electrons and having a larger format CCD that is significantly better suited for imaging at 200-300keV. The equipment will be attached to our pre-existing Philips 300keV electron microscope in the Electron Microscope Laboratory of the Department of Biochemistry at UC San Francisco. This facility is available to the entire campus as well as to outside users. All of the core users except two are currently funded by the NIH, and all have extensive experience in electron microscope. The core users plus the supervisor of the EM lab will form an advisory committee to oversee use of the instrument and to set policy. Use by other regional and national users will be encouraged in the time remaining after core investigator use. UC San Francisco will provide space and staff support (including software) for operating the equipment.