OLYMPUS DSU CONFOCAL SYSTEM: DROSOPHILIA, C ELEGANS, & MOUSE

该子项目是利用由NIH/NCRR资助的共享仪器补助金提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是资助机构，不一定是研究者的机构。描述（由申请人提供）：获得高分辨率的活细胞图像和电影的能力对于理解生物过程以及它们如何受到疾病或突变的干扰至关重要。此提案是购买奥林巴斯光盘扫描单元（DSU）共聚焦显微镜系统。与激光扫描共聚焦显微镜不同，奥林巴斯DSU系统使用白色光源，从而大大降低了系统的初始成本和维护。为了弥补来自白光光源的较低光强度，奥林巴斯DSU系统使用高灵敏度的冷却电荷耦合器件（CCD）相机来捕获荧光信号。失焦光被旋转的圆盘消除，圆盘上有精确定义的狭缝，形成虚拟针孔照明。快速图像采集（高达15帧/秒）和降低旋转光盘照明的光毒性相结合，使固定细胞和活细胞的高质量成像成为可能。奥林巴斯DSU系统将被安置在遗传学系，由NIH R01资助的各种项目的研究人员使用。这些包括果蝇组织中数百种gfp融合蛋白的成像（Cooley），小鼠和秀丽隐杆线虫种系细胞中的抗体定位（Reinke, Sweasy），秀丽隐杆线虫的细胞迁移（Stern），配体处理细胞中PDGF受体突变形式的定位（DiMaio），培养细胞中的磷酸肌苷信号传导（H. Sun），以及发育中的斑马鱼肾脏的实时成像（Z. Sun）。奥林巴斯DSU系统将取代两台过时的非电动荧光显微镜，这些显微镜不能再为这些项目提供必要的数据。它还将为昂贵的激光扫描共聚焦成像提供急需的替代方案，特别是在荧光信号强度不受限制的情况下。此外，与基于激光的系统相比，该系统将允许随时使用延时显微镜，再次降低成本。

This subproject is one of many research subprojects utilizing the resources provided by a Shared Instrumentation Grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the grant, which is not necessarily the institution for the investigator. DESCRIPTION (provided by applicant): The ability to obtain high-resolution, live-cell images and movies is essential for understanding biological processes and how they are perturbed by disease or mutation. This proposal is for the purchase of an Olympus Disc Scan Unit (DSU) confocal microscope system. Unlike laser-scanning confocal microscopy, the Olympus DSU system uses a white light source, thus dramatically reducing the initial cost and maintenance of the system. To compensate for the lower light intensity available from white-light sources, the Olympus DSU system uses a highly sensitive cooled charged-couple device (CCD) camera to capture fluorescence signals. Out-of-focus light is eliminated by the spinning disc with precisely defined slits that create virtual pin-hole illumination. Fast image acquistion (up to 15 frames/sec) and lowered phototoxicity from spinning disc illumination combine to allow high-quality imaging of both fixed and live cells. The Olympus DSU system will be housed in the Department of Genetics and used by investigators for a variety of projects funded by NIH R01 grants. These include imaging of hundreds of GFP-fusion proteins in Drosophila tissues (Cooley), antibody localization in mouse and C. elegans germline cells (Reinke, Sweasy), cell migration in C. elegans (Stern), localization of mutant forms of PDGF receptor in cells treated with ligands (DiMaio), phosphoinositide signaling in cultured cells (H. Sun), and live imaging of developing zebrafish kidneys (Z. Sun). The Olympus DSU system will replace two out-dated, non-motorized fluorescence microscopes that can no longer produce data essential for these projects. It will also provide a much-needed alternative to expensive laser-scanning confocal imaging, especially in cases where the intensity of fluorescence signals is not limiting. Furthermore, the system will allow ready access to time-lapse microscopy, again with a reduced cost compared to laser-based systems.