IDBR: Development of a High Resolution Serial Block-Face Scanning Electron Microscope

神经网络的详细电路图及其神经元之间的突触连接是推进中枢神经系统研究的必要条件。这种需求在新兴的“连接组学”领域尤为突出，该领域需要大量高分辨率三维显微镜宽视场图像来可视化和分析中枢神经系统的神经回路。聚焦离子束连续块面扫描电子显微镜（FIB-SBFSEM）和连续切片透射电子显微镜（system）等仪器可以实现高分辨率的三维图像，并提供追踪精细树突和突触所需的分辨率。然而，串行块脸扫描电子显微镜（SBFSEM）是唯一的三维显微镜技术，可以提供宽视场图像。目前迫切需要一种能够提供高分辨率三维宽视场图像的单一仪器。首席研究员（PI）将与JEOL公司合作，采取关键的第一步，通过改进透镜（TTL）背散射探测器来满足这一需求，这将提高背散射电子图像（BEI）分辨率并提高图像采集速度。智力优势：TTL后向散射检测器的改进将极大地提高神经科学家的能力：1)识别特定的脊髓神经元表型；2)深入了解中枢神经系统区域的“难以穿透的丛林”，从而克服100多年前发现的障碍。更广泛的影响：TTL后向散射检测器的改进将：1)允许绘制中枢神经系统某些区域的基本突触微电路；2)用于培养连接组学新兴领域的下一代西班牙裔科学家；3)满足30名波多黎各神经科学家使用仪器的需求；在beta测试成功完成后，随着开发和市场推广，该仪器将满足许多从事连接和其他中枢神经系统研究的神经科学家的需求。传播：PI将为西班牙裔神经生物学研究生和30名神经生物学教员提供有关新仪器的培训。PI的实验室将其研究图像提供给在其小册子中使用它们的行业，适当地归功于PI和资助代理。这种安排将继续并扩大。TTL反向散射探测器将创造机会，利用令人惊叹的视觉图像，作为提高对科学新发展的认识的一种方式。为了实现这一目标，我们将：1)使用这些项目生成的图像作为UPR-RP课堂和会议室的艺术品；2)向UPR-RP校友杂志和Inventio提交文章，Inventio是一份双语校园出版物，向更大的商业和工业社区以及公众展示研究和学术成果；3)利用这些图片说服当地报纸和杂志多报道基础科学。我们希望用扣人心弦和美丽的图像来捕捉公众的想象力和兴趣。最后，通过与加州大学洛杉矶分校（UCLA）神经成像实验室（LONI）的小鼠生物医学成像研究网络（小鼠BIRN）的合作，我们使用了小鼠BIRN绘图工具包（MBAT），这是一个独立于平台的图像查看器，能够在单个查看“画布”上同时呈现来自多个来源的多个图像。使用MBAT，本地和在线图像可以并排比较和探索。MBAT允许几乎任何拥有普通个人电脑和互联网连接的人在我们的网站（http://pisces.cnnet.clu.edu-lab/final/）上查看我们图像数据的虚拟部分。

Detailed circuit maps of neural networks and their synaptic connections between/among neurons of the central nervous system (CNS) are needed to advance CNS studies. This need is particularly acute in the emerging field of "connectomics" that requires a very large number of high-resolution three-dimensional microscopy wide-field images to visualize and analyze CNS neural circuitry. Instruments such as Focused Ion Beam Serial Block Face Scanning Electron Microscopy (FIB-SBFSEM) and serial section transmission electron microscopy (ssTEM) achieve high-resolution three-dimensional images and provide the resolution required to trace fine dendrites and synapses. However, Serial Block Face Scanning Electron Microscopy (SBFSEM) is the only three-dimensional microscopy technique that can provide wide-field images. A single instrument that provides high-resolution three-dimensional wide-field images is acutely needed. The principal investigator (PI) in concert with JEOL Inc. will take the first crucial step to meet that need by improving a thru-the-lens (TTL) backscatter detector that will increase the backscattered electron image (BEI) resolution and increase image acquisition speed. Intellectual merit: Improvements in the TTL backscatter detector will greatly advance neuroscientists' ability to 1) identify specific spinal neuron phenotypes and 2) gain insights into the 'impenetrable jungles' of central nervous system regions, thus overcoming obstacles identified over 100 years ago. Broader Impacts: TTL backscatter detector improvements will: 1) permit mapping of basic synaptic microcircuits of some regions of the central nervous system; 2) be used to train next generation Hispanic scientists in the emerging field of connectomics; 3) meet 30 Puerto Rican neuroscientists' need for access to instrumentation; upon successful completion of the beta test and with development and marketing, the instrument will 4) meet the need of many, many neuroscientists engaged in connectivity and other CNS studies. Dissemination: The PI will offer Hispanic neurobiology graduate students and 30 neurobiology faculty members training on the new instrument. The PI's laboratory provides its research images to industry which uses them in its brochures, appropriately crediting the PI and funding agents. This arrangement will continue and expand. The TTL backscatter detector will create opportunities to exploit stunning visual images as a way of enhancing awareness of new developments in science. Towards this goal we will: 1) use images generated from these projects as artwork in UPR-RP class and conference rooms; 2) submit articles to the UPR-RP alumni magazine and to Inventio, a bi-lingual campus publication that presents research and scholarly achievements to the larger business and industry community and to the general public; and 3) use the images to persuade local newspapers and magazines to do more stories on basic science. We hope to use riveting and beautiful images to capture the imagination and interest of the lay public. Finally, through a collaboration with the mouse Biomedical Imaging Research Network (mouse BIRN) in the Laboratory of Neuro Imaging (LONI) at University of California Los Angeles (UCLA), we use the Mouse BIRN Atlasing Toolkit (MBAT), a platform independent image viewer with the ability to simultaneously present multiple images from multiples sources on a single viewing "canvas". With MBAT, local and online images can be compared and explored side-by-side. MBAT allows almost anyone with a modest personal computer and internet connection to view virtual sections of our image data on our website (http://pisces.cnnet.clu.edu-lab/final/).