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

描述(由申请人提供)：我们要求一台双光束扫描电子显微镜(SEM)，这将被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目前提供最先进的仪器和相关的技术支持，通过透射电子显微镜(TEM)、核磁共振和X射线衍射对大分子的结构进行分析。这些设施由十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、纽约城市大学、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、卫生部沃兹沃斯中心、康奈尔大学威尔医学院和纽约州立大学。增加双光束扫描电子显微镜的主要目标是扩大NYSBC可以解决的项目范围，特别是为通常依赖光学显微镜对组织进行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查看成像模式，在该模式下，交替对树脂嵌入的组织块的表面进行成像和蚀刻，以生成贯穿组织的一系列连续的2D图像。然后，通过使用专用软件对准和堆叠这些图像，获得5-40 nm分辨率的细胞内容的3D渲染。另一个目标是使用扫描透射式(STEM)探测器来分析大分子络合物的质量分布，从而便于使用透射电子显微镜成像和单粒子分析来确定结构。此外，我们将在NYSBC的两个开发项目中使用双光束扫描电子显微镜；即制造用于透射电子显微镜成像的相位板和冷冻水合细胞培养物的减薄，以制造适合用于冷冻电子断层扫描的样品。描述了13个单独的用户项目，代表了将从这项技术中受益的具体生物应用。这些项目包括四个神经生物学项目，两个涉及细菌和艾滋病毒发病机制的项目，以及涉及自噬、内吞、低密度脂蛋白加工和细胞与细胞黏附的细胞结构研究。此外，还描述了正在进行的负责DNA和RNA复制和核运输的大分子络合物的透射电子显微镜研究，这将从STEM分析和相板开发中受益。这些研究人员来自六个不同的纽约机构，他们代表了在NYSBC实施双光束扫描电子显微镜所产生的研究活动的样本。通过添加双光束扫描电子显微镜提供的新技术，这些新技术已经在NYSBC得到支持，我们将显著提高其作为大纽约大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.