Upgrade Existing Multiwell, Fluorescence Timelapse Imaging Workstation to Confoca

描述(申请人提供)：我们最初的荧光延时系统购买于2001年(共享仪器授权1 S10 RR15768-01，“活细胞和组织的荧光成像”，首席研究员：穆勒)。迫切需要升级，以满足NIH资助的9名研究人员的核心对多色、多井、时间流逝成像的要求。虽然变形软件和计算机已经使用隆巴迪综合癌症中心基金(LCCC)升级过一次，而且乔治敦大学的两名研究人员(穆勒和埃尔门多夫)在2006年联合购买了筛查软件模块，但该仪器不是共焦的。多孔自动成像功能使数小时或数天的多个平行实验成为可能，并极大地提高了研究效率和在相同培养条件下比较治疗方式的能力。过去的应用包括细胞迁移分析、长期成像以记录肿瘤细胞行为的变化或GFP嵌合蛋白的亚细胞定位，以及药物筛选。该系统包括一台尼康Eclipse TE300倒置表观荧光显微镜，ORCA-ER单色冷却CCD12位相机，以及带有Multiwell筛选和分析软件模块的变形图像采集和分析软件版本7，一台Sutter Instruments DG-4用于多色实时成像的高速波长切换器，Preor ProScan电动x，y，z舞台控制器，亮场和荧光快门和滤光轮，以及一个环境室。我们要求资金将这个系统转换为带有二极管激光的共焦系统，一个用于提高灵敏度的EM-CCD相机，以及一个快速发射滤光轮。从2004年到2007年，18名调查人员使用该仪器超过2230小时。已经发表了四篇论文，两篇已提交，几篇正在利用这一显微镜系统进行准备。它存放在隆巴迪综合癌症中心(LCCC)的显微镜和成像共享资源中，该中心由肿瘤学教授米勒博士指导。这家医院的经理是布莱恩·科斯博士。徐博士和戈文丹博士分别通过指导TIRF/多光子组件和教育/培训参与其中。该机构由长期合作伙伴关系、按存储容量使用计费费用和长期合作伙伴关系癌症中心支持补助金(2P30-CA-51008)提供资金。公共卫生相关性：这项资金购买激光共聚焦升级到研究显微镜的提案将主要支持文森特·T·隆巴迪癌症中心、指定的NIH综合癌症中心以及乔治敦大学医学中心和主校区的其他研究人员的研究。这种激光共聚焦升级将有助于识别可能导致肿瘤细胞侵袭和血管生成、高血压、真菌和疟疾感染以及免疫发展的蛋白质和亚细胞结构的细胞位置，以及有效的药物靶向。这台显微镜将被放置在一个预先存在的共享资源中，由隆巴迪的一个核心调查人员使用，并提供给其他同样得到NIH资助的拨款支持的乔治敦大学的调查人员。

DESCRIPTION (provided by applicant): Our original epifluorescence timelapse system was purchased in 2001 (Shared Instrumentation grant 1 S10 RR15768-01, "Fluorescence Imaging of Live Cells and Tissues", principal investigator: Mueller). It is critical need of upgrading to fulfill requirements for multicolor, multiwell, timelapse imaging for a core of nine NIH funded investigators. Although the Metamorph software and computer have been upgraded once using Lombardi Comprehensive Cancer Center Funds (LCCC), and a screening software module was purchased jointly by two Georgetown investigators in 2006 (Mueller and Elmendorf), this instrument is not confocal. The multiwell, automated imaging feature makes possible multiple, parallel experiments over hours or days and dramatically increases research efficiency and the power to compare treatment modalities under identical culture conditions. Past applications include cell migration assays, long term imaging to document changes in tumor cell behavior or in subcellular localization of GFP-chimeric proteins, and drug screening. The system consists of a Nikon Eclipse TE300 inverted epi-fluorescence microscope, ORCA-ER monochrome cooled CCD 12-bit camera, and MetaMorph image acquisition and analysis software version 7 with the Multiwell Screening and Analysis software module, a Sutter Instruments DG-4 high speed wavelength switcher for multi-color, live imaging, Prior ProScan motorized x,y,z stage controller, bright field and fluorescence shutters and filter wheels, and an environmental chamber. We request funds to convert this system to a confocal system with diode lasers, an EM-CCD camera for increased sensitivity, and a fast emission filter wheel. From 2004-2007, 18 investigators have utilized this instrument over 2230 hours. Four papers have been published, two submitted, and several are in preparation utilizing this microscope system. It is housed in the Microscopy and Imaging Shared Resource of the Lombardi Comprehensive Cancer Center (LCCC) which is directed by Dr. Mueller, a Professor of Oncology. The manager for this facility is Dr. Brian Koss. Drs. Xu and Govindan participate by directing the TIRF/ multiphoton component and education/ training, respectively. This facility is funded by LCCC, chargeback fees, and the LCCC Cancer Center Support Grant (2P30-CA-51008). PUBLIC HEALTH RELEVANCE: This proposal for funds to purchase a laser confocal upgrade to a research microscope will primarily support research at the Vincent T. Lombardi Cancer Center, a designated NIH Comprehensive Cancer Center and other investigators in the Georgetown University Medical Center and Main Campus. This laser confocal upgrade will facilitate the identification of cellular locations of proteins and subcellular structures that may cause tumor cell invasion and angiogenesis, development of hypertension, fungal and malarial infections and development of immunity, and effective drug targeting. This microscope will be placed in a pre-existing shared resource, used by a core of Lombardi investigators, and made available to other Georgetown investigators also supported by NIH-funded grants.