OLYMPUS DSU CONFOCAL SYSTEM: ZEBRAFISH:POLYCYSTIC KIDNEY DISEASE

这个子项目是利用由NIH/NCRR资助的共享仪器赠款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是用于拨款的，而不一定是用于调查人员的机构。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了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。这项提案是为了购买奥林巴斯圆盘扫描单元(DSU)共焦显微镜系统。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，从而极大地降低了系统的初始成本和维护。为了补偿白光光源提供的较低的光强度，奥林巴斯的DSU系统使用了一个高灵敏度的冷却电荷耦合器件(CCD)相机来捕获荧光信号。旋转的圆盘具有精确定义的狭缝，从而产生虚拟针孔照明，从而消除了失焦的光。快速的图像采集(高达15帧/秒)和旋转光盘照明降低的光毒性相结合，使固定细胞和活细胞都能获得高质量的成像。奥林巴斯的DSU系统将被安置在遗传学系，并由研究人员用于由NIH R01拨款资助的各种项目。这些包括果蝇组织中数百个GFP融合蛋白的成像(Cooley)，抗体在小鼠和线虫胚系细胞中的定位(Reinke，Sweasy)，线虫中的细胞迁移(Stern)，PDGF受体突变形式在经配体处理的细胞中的定位(DiMaio)，培养细胞中的磷脂酰肌醇信号(H.Sun)，以及发育中的斑马鱼肾脏的实时成像(Z.Sun)。奥林巴斯的DSU系统将取代两台过时的非机动荧光显微镜，这两台显微镜不再能产生这些项目所需的数据。它还将为昂贵的激光扫描共聚焦成像提供一种急需的替代方案，特别是在荧光信号强度不受限制的情况下。此外，该系统将允许随时访问延时显微镜，与基于激光的系统相比，成本也更低。

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.