JEOL JEM-1400 Tomographic Transmission EM with High-Resolution Digital Cameras

描述(由申请人提供)：来自科罗拉多州立大学三个系的五名现有研究员和三名NIH资助的新研究员，加上来自怀俄明大学的一名新研究员，以及一名来自阿尔伯特·爱因斯坦医学院的分包研究员，申请804,260美元的NCRR资金，以购买一台JEOL JEM-1400 120千伏透射电子显微镜，配备一个液氮冷却的700层析标本倾斜支架、一台快扫描数码相机和一台高分辨率慢扫描数码相机。新的瞬变电子显微镜将取代已有19年历史的JEOL JEM-2000 EX-II 200千伏“主力”瞬变电子显微镜，它没有数码相机，没有层析成像能力，也没有自动舞台驱动和自动摄影能力。它的“剪胶片”相机已经制作了67,000张底片(&gt；67,000个机械循环)，需要我们购买三个新的照相盒，因为磨损的机械推进器经常卡住，无法挽回地损坏三个。在过去五年中，JEM-2000的维修频率不断增加(“停机时间”为20-38个工作日)。所要求的底部安装式高分辨率数码相机，与自动倾斜和自动摄影相结合，对于断层分析(每次重建最多70张图像)是必不可少的，将极大地促进传统摄影。数字图像将集中存储，以供在线或离线访问，而不可能更改或删除已存档的原件。在群体分析过程中，视频速率为11Mb的相机将有助于在监视器上聚焦和快速跟踪图像细节，并将成为获得常规测量显微照片的主要相机。使用4K(16MB)高分辨率数码相机(而不是2K或1K相机)捕获数百个病毒颗粒的单一图像，将使断层分析所需的曝光次数减少4倍或16倍，从而极大地减少光束对冷冻薄膜中病毒/受体-蛋白质相互作用的损害。在线立体成像通常是在极端倾斜的情况下进行的，对于解释冰冻断裂复制品中的表面形貌和免疫金标记物的“侧面”是必要的，而不是等到第二天胶片冲洗出来。消除实验之间的这种延迟将极大地加快几个实验室的工作。目前“主要用户”的高生产率已导致&gt；1100份来自JEM-2000的电子显微照片在&gt；50份同行评议出版物中发表。对于NIH资助的6个主要用户来说，EM是必不可少的，这些用户研究了神经元和神经胶质膜的分子组成(RASH)、神经退行性疾病中的ADF/粘连蛋白-肌动蛋白棒(Bamburg)、神经元传递的突触和非突触机制(Reist；Rash)、病毒/蛋白质分子相互作用(Hafenstein)、K+通道功能和运输的调节因子(Tamkun)以及神经元缝隙连接的可塑性(PEREDA/RASH)。根据19年使用JEM-2000电子显微镜的经验，一名占10%时间的行政和技术人员(Davidson女士)和一个基础广泛的咨询委员会将确保升级后的更新换代电子显微镜的持续高效调度和操作。

DESCRIPTION (provided by applicant): Five established and three new NIH-funded investigators from three departments at Colorado State University, plus one new investigator from the University of Wyoming, and one established investigator via a subcontract from Albert Einstein College of Medicine, request $804,260 in NCRR funds to purchase a JEOL JEM-1400 120 kV transmission electron microscope (TEM) equipped with a liquid nitrogen-cooled 700 tomographic specimen tilt holder, a fast-scan digital camera, and a high-resolution slow-scan digital camera. The new TEM will replace a 19-yr-old JEOL JEM-2000 EX-II 200 kV "workhorse" TEM that lacks a digital camera, has no tomographic capability, and has no automated stage drives and no automated photographic capability. Its "cut- film" camera has made >67,000 negatives (>67,000 mechanical cycles), requiring us to purchase three new camera boxes because the worn mechanical advance frequently jammed, irrevocably damaging three. The JEM-2000 has had increasing frequency of repairs ("down time" of 20-38 working days in each of the last five years. The requested bottom-mount high-resolution digital cameras, in combination with automated tilting and automated photography, are essential for tomographic analysis (up to 70 images for each reconstruction) and will greatly facilitate conventional photography. Digital images will be stored centrally for access online or offline without possibility of altering or deleting the archived originals. The video-rate 11MB camera will facilitate focusing and rapid tracking of image detail on the monitor during group analysis, and will be the primary camera for obtaining routine survey micrographs. Single-image capture of hundreds of virus particles using the 4K (16MB) high-resolution digital camera (as opposed to 2K or 1K cameras) will greatly reduce beam damage to virus/receptor-protein interactions in frozen thin films by reducing by 4x or 16x the number of exposures needed for tomographic analysis. On-line stereoscopic imaging, often at extreme tilts, is a necessity for interpreting surface topography and the "sidedness" of immunogold-labels in freeze-fracture replicas, rather than waiting until the next day for film to be developed. Removal of this delay between experiments will greatly accelerate work in several laboratories. The high productivity of current "major users" has resulted in >1100 published electron micrographs from the JEM-2000 in >50 peer-reviewed publications. TEM is essential for 6 NIH-funded major users investigating molecular composition of neuronal and glial membranes (Rash), ADF/cofilin-actin rods in neurodegenerative disease (Bamburg), synaptic and non-synaptic mechanisms of neuronal transmission (Reist; Rash), virus/protein molecular interactions (Hafenstein), regulators of K+ channel function and trafficking (Tamkun), and plasticity of neuronal gap junctions (Pereda/Rash). Based on 19 years of experience with the JEM-2000 TEM, a 10%-time administrative and technical person (Ms. Davidson) and a broadly-based Advisory Board will ensure continued efficient scheduling and operation of the upgraded replacement transmission electron microscope.