Serial Block Face Scanning Electron Microscope

 描述(申请人提供)：我们建议将三维电子显微镜(3D EM)添加到我们的EM共享资源中。我们已经提供了40年的显微镜服务，是校园里持续运行时间最长的共享资源。许多用户需要在超微结构水平上进行3D重建。目前，杜克大学可以通过共聚焦显微镜进行3D分析，但它只能提供光学显微镜分辨率；使用商业3D EM实验室成本高昂。增加3D EM能力将节省赠款资金并减少周转时间。使用连续块面部扫描电子显微镜(SBFSEM)，可以高分辨率、自动和快速地对大面积(细胞与细胞之间的关系)进行成像，并在1-2天内获得重建数据。传统上，超微结构三维重建一直是 通过连续的超薄切片并在许多网格上找到相同的区域-这是一项乏味和耗时的工作(数月)。可供选择的程序包括电子断层扫描(ET)(但截面厚度和倾斜量有限)和聚焦离子束(FIB)显微镜(面积和深度也有限)，这两种方法在这里都不提供。自动胶带收集超显微切割机(ATUM)具有保存切片以供重新检查的优势，但需要保持与每个切片保持一致，并且在扫描电子显微镜下存在起皱和对齐问题。因此，我们相信SBFSEM最适合我们调查人员的不同需求。SBFSEM对这里的所有调查人员来说都是必不可少的。其中两个已经发布了3D EM数据；有几个已经收到了我们准备的材料的3D数据，并在测试设施中进行了成像。另一位花了几个月时间做连续切片和常规传输EM的人收到了我们获得的SBFSEM数据。另外两个用户有服从ET的项目(此处不可用)，但可能通过SBFSEM；所有其他用户都具有共焦或MRI 3D经验，需要3D EM。所有17个项目都是关于确定疾病标志物或治疗目标。我们的建议是可行的。我们得到了杜克医学院(院长南希·安德鲁斯)和病理系(大卫·豪厄尔博士)的支持，并为太空提供了运营资金和病理系。我们通过Core为活体显微镜(CVIM；Allan Johnson博士)、光学显微镜核心设备(LMCF；Sam Johnson博士)、CoreResearch@Duke(Iain Sanderson博士)、Duke Health Technology Systems(比利·威利斯博士)和Duke研究计算办公室(Mark DeLong博士)提供充足的数据存储和分析能力。我们有很长的服务历史；准备样本、运行仪器、收集数据集并将其呈现为3D的技术能力(Mark Ellisman博士)；以及表明该提议可持续的5年财务计划。请参阅我们的支持信。最后，从长远来看 为了保持稳定，我们已经确定了一位较年轻的同事，在下一个十年后担任资源总监。

 DESCRIPTION (provided by applicant): We propose to add Three-Dimensional Electron Microscopy (3D EM) to our EM Shared Resource. We have provided microscopy service for 4 decades and are the longest continuously-running Shared Resource on campus. Many users need 3D reconstructions at the ultrastructural level. Currently, 3D analysis is attainable at Duke by confocal microscopy, but it gives only light microscopy resolution; using a commercial 3D EM laboratory is expensive. Addition of 3D EM capability would save grant funds and decrease turn-around time. With a Serial Block Face Scanning Electron Microscope (SBFSEM) large areas (cell-to-cell relationships), can be imaged at high resolution, automatically, and rapidly, with daa available in 1-2 days for reconstruction. Traditionally, ultrastructural 3D reconstruction has been done by serial ultrathin sectioning and finding the same area on many grids-a tedious and time-consuming job (months). Alternative procedures include Electron Tomography (ET) (but section thickness and amount of tilt are limited) and Focused Ion Beam (FIB) Microscopy (where area and depth are also limited), neither of which is available here. The Automated Tape-collecting UltraMicrotome (ATUM) has the advantage of preserving sections for reexamination, but requires keeping up with each of them and has issues of wrinkling and alignment in the SEM. Thus, we believe SBFSEM would best fit the varying needs of our investigators. SBFSEM is essential for all investigators herein. Two have already published 3D EM data; several have received 3D data on material we prepared and imaged at test facilities. Another, who spent months doing serial sectioning and routine transmission EM, has received SBFSEM data that we obtained. Two additional users have projects amenable to ET (unavailable here), but possible by SBFSEM; all others have confocal or MRI 3D experience and need 3D EM. All 17 projects are on identifying disease markers or treatment targets. Our proposal is viable. We have support of the Duke School of Medicine (Dean Nancy Andrews) and the Department of Pathology (Dr. David Howell) with operating funds and the Department of Pathology for space. We have ample data storage and analysis capability through the Core for In Vivo Microscopy (CVIM; Dr. Allan Johnson), the Light Microscopy Core Facility (LMCF; Dr. Sam Johnson), the CoreResearch@Duke (Dr. Iain Sanderson), the Duke Health Technology Systems (Dr. Billy Willis), and the Office of Duke Research Computing (Dr. Mark DeLong). We have a long history of service; the technical ability to prepare specimens, run the instrument, and collect and render the data sets to 3D (Dr. Mark Ellisman); and a 5-year Financial Plan showing that the proposal is sustainable. Please see our letters of support. Finally, for long-term stability, we have identified a younger colleague to assume the resource directorship after the next decade.