UltraViewRS Spinning Disk Confocal Microscopy

描述(由申请人提供)：这项提案解决了位于怀特黑德研究所内的W.M.Keck生物成像设施对更多共焦显微镜能力和能力的需求。这一核心设施在1998至2003年间为20个怀特黑德实验室、39个麻省理工学院实验室和4个其他机构的实验室提供协调一致的培训和服务，总计使用3732小时。现有的ZeissLSM510-Meta基于点扫描技术，因此从根本上限制了图像采集速度。在评估了几种相互竞争的技术后，我们确定Perkin Elmer的第二代旋转磁盘系统UltraViewRS最合适。这些技术将解决对额外共焦容量的需求以及成像快速生物过程的能力。该系统将允许用户以现有LSM510的10倍以上的速度收集时间分辨率的2D或3D共焦数据。虽然LSM510-Meta仍将是该机构的核心技术，特别适用于对厚厚的固定组织样本进行成像，但UltraViewRS提供的补充能力将使更多和更广泛的重要生物学研究得以进行。在项目描述部分，我们描述了7个怀特黑德实验室和3个麻省理工学院实验室已经在进行的需要这些增强的共焦能力的研究。这些项目包括研究细胞运动过程中的细胞骨架结构、胚胎发育、微生物发病机制、蛋白质构象疾病、衰老、膜动力学和红细胞生成。拟议的系统将允许激发相关的活细胞探针，包括所有主要的绿色荧光蛋白变体以及常用的化学探针和固定细胞荧光团。除了提高数据收集的速度外，拟议的系统还通过减少样本光损伤而使用户受益，这对研究胚胎发育的研究人员特别有用。另外两个关键系统部件--环境室和机动XY工作台--将为该设施提供急需的活细胞能力，并通过自动化多点数据收集制度使仪器得以有效使用。怀特黑德研究所在维护和进一步投资NIH资助的共享仪器方面有着良好的记录，W.M.凯克生物成像设施内资源的不断增加就证明了这一点。

DESCRIPTION (provided by applicant): This proposal addresses the need for additional confocal microscopy capacity and capability at the W.M. Keck Biological Imaging Facility located within the Whitehead Institute. This core facility provides confocal training and service to 20 Whitehead labs, 39 from MIT and 4 from other institutions totaling 3732 hours of use between 1998 and 2003. The existing ZeissLSM510-Meta is based on point-scanning technology and is thus fundamentally limited in image acquisition speed. After evaluating several competing technologies that would address the need for additional confocal capacity as well as the ability to image fast biological processes, we determined that the second generation spinning-disk system from Perkin Elmer, the UltraViewRS, was most appropriate. This system will allow users to collect time-resolved 2D or 3D confocal data at over 10x the rate of the existing LSM510. While the LSM510-Meta will remain a core technology within the Facility, especially suited to imaging thick fixed tissue samples, the complementary capability provided by the UltraViewRS will enable an additional and broad range of important biological investigations to be undertaken. In the project descriptions section, we have described studies already underway in 7 Whitehead and 3 MIT laboratories that require these enhanced confocal capabilities. These projects include investigation of cytoskeletal structure during cell motility, embryonic development, microbial pathogenesis, protein conformational diseases, senescence, membrane dynamics, and erythropoiesis. The proposed system will allow excitation of relevant live-cell probes including all major GFP variants as well as commonly-used chemical probes and fixed-cell fluorophores. Apart from the enhanced speed of data collection, the proposed system also benefits users via reduced sample photo-damage, which is especially useful for those investigators studying embryonic development. Two other key system components, an environmental chamber and a motorized XY-stage, will provide much needed live-cell capabilities to the Facility and allow efficient use of the instrument through automated multi-point data collection regimes. The Whitehead Institute has an excellent track record of maintaining and further investing in NIH-funded shared instrumentation as documented in the ever-increasing use of the resources within the W.M. Keck Biological Imaging Facility.