Dual-Beam Scanning Electron Microscope for New York Structural Biology Center

描述（由申请人提供）：我们正在申请一台双光束扫描电子显微镜（SEM），该显微镜将添加到纽约结构生物学中心（NYSBC）的共享电子显微镜设施中。NYSBC目前通过透射电子显微镜（TEM）、NMR和X射线衍射为大分子结构分析提供最先进的仪器和相关技术支持。这些设施由10个纽约学术机构共享，其中大部分位于纽约市：阿尔伯特·爱因斯坦医学院、纽约、哥伦比亚大学、纪念斯隆-凯特琳癌症中心、西奈山医学院、纽约大学、洛克菲勒大学、沃兹沃斯中心卫生系、康奈尔大学威尔医学院、和纽约的州立大学。增加双光束SEM的主要目标是扩大NYSBC可以解决的项目范围，特别是为通常依赖光学显微镜进行组织形态评估的细胞生物学家和神经生物学家提供更高分辨率的替代方案。为了实现这一目标，我们将实施自动切片和视图成像模式，其中树脂包埋的组织块的表面交替成像和蚀刻，以生成通过组织的连续系列的2D图像。然后通过使用专用软件对齐和堆叠这些图像来获得5-40 nm分辨率的细胞内容物的3D渲染。另一个目标是使用扫描透射（STEM）检测器来分析大分子复合物的质量分布，从而促进使用TEM成像和单粒子分析的结构确定。此外，我们将使用双束扫描电镜在NYSBC的两个开发项目;即，TEM成像相板的制造和冷冻水化细胞培养物的细化，以制造合适的样品冷冻电子断层扫描。十三个单独的用户项目进行了描述，代表特定的生物应用，将受益于这项技术。其中包括四个神经生物学项目，两个项目涉及细菌和艾滋病毒的发病机制，以及涉及自噬，内吞作用，LDL加工和细胞-细胞粘附的细胞结构的研究。此外，正在进行的TEM研究负责DNA和RNA复制和核运输的大分子复合物的描述，这将受益于干分析和相位板的发展。这些研究人员来自六个不同的纽约机构，并代表了一个抽样的研究活动，将导致在NYSBC实施双光束扫描电镜。通过增加双光束扫描电镜提供的新技术，我们将大大提高其作为大纽约都市区结构生物学中心的地位。

DESCRIPTION (provided by applicant): We are requesting a dual-beam scanning electron microscope (SEM) that will be added to the shared electron microscopy facility at the New York Structural Biology Center (NYSBC). NYSBC currently provides state-of- the-art instrumentation and associated technical support for structural analysis of macromolecules by transmission electron microscopy (TEM), NMR and X-ray diffraction. These facilities are shared by a group of ten New York academic institutions, most of which are in New York City: Albert Einstein College of Medicine, City University of New York, Columbia University, Memorial Sloan-Kettering Cancer Center, Mount Sinai School of Medicine, New York University, The Rockefeller University, Wadsworth Center of the Department of Health, Weill Medical College of Cornell University, and the State University of New York. The primary goal of adding a dual-beam SEM is to expand the range of projects that can be addressed at NYSBC and specifically to offer a higher resolution alternative to cell biologists and neurobiologists who typically rely on light microscopy for morphological evaluation of tissue. To achieve this goal, we will implement an automated slice- and-view imaging mode, whereby the surface of a resin-embedded block of tissue is alternately imaged and etched, to generate a sequential series of 2D images through the tissue. A 3D rendering of the cell contents at 5-40 nm resolution is then obtained by using dedicated software to align and stack these images. An additional goal is to use a scanning transmission (STEM) detector to analyze the mass distribution of macromolecular complexes, thus facilitating structure determination using TEM imaging and single particle analysis. Additionally, we will use the dual-beam SEM for two development projects at NYSBC; namely, the manufacture of phase plates for TEM imaging and the thinning of frozen-hydrated cell cultures in order to fabricate suitable samples for cryoelectron tomography. Thirteen individual user projects are described, representing specific biological applications that will benefit from this technology. These include four neurobiology projects, two projects addressing pathogenesis of bacteria and HIV, and studies of cellular structures involved in autophagy, endocytosis, LDL processing and cell-cell adhesion. In addition, ongoing TEM studies of macromolecular complexes responsible for DNA and RNA replication and nuclear transport are described, which would benefit from STEM analysis and phase plate development. These researchers come from six different New York institutions and represent a sampling of research activities that would result from implementation of dual-beam SEM at NYSBC. By adding the new technologies provided by dual-beam SEM those already supported at NYSBC, we will significantly enhance its standing as a hub of structural biology in the greater New York metropolitan area.