Upgrade Existing Multiwell, Fluorescence Timelapse Imaging Workstation to Confoca

描述（由申请人提供）：我们最初的落射荧光时间流逝系统于2001年购买（共享仪器授权1 S10 RR 15768 -01，“活细胞和组织的荧光成像”，主要研究者：Mueller）。这是迫切需要升级，以满足要求，为核心的9个国家卫生研究院资助的研究人员的双稳态，多孔，时间推移成像。虽然Metamorph软件和计算机已经使用Lombardi综合癌症中心基金（LCCC）进行了一次升级，并且2006年乔治敦的两位研究人员（Mueller和Elmendorf）联合购买了一个筛查软件模块，但该仪器不是共焦的。多孔自动成像功能使多个平行实验在数小时或数天内成为可能，并大大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或GFP嵌合蛋白亚细胞定位的变化，以及药物筛选。该系统由Nikon Eclipse TE 300倒置落射荧光显微镜、ORCA-ER单色冷却CCD 12位相机和MetaMorph图像采集和分析软件版本7（具有多孔筛选和分析软件模块）、用于多色、实时成像的Sutter Instruments DG-4高速波长切换器、Prior ProScan电动x、y、z载物台控制器、明场和荧光快门和滤光轮，以及环境室。我们要求资金将该系统转换为具有二极管激光器的共焦系统，用于提高灵敏度的EM-CCD相机和快速发射滤光轮。从2004年至2007年，18名调查员使用这一工具超过2230小时。四篇论文已发表，两个提交，和几个正在准备利用这种显微镜系统。它位于Lombardi综合癌症中心（LCCC）的显微镜和成像共享资源中，由肿瘤学教授Mueller博士指导。这个机构的经理是布莱恩·科斯博士。Xu博士和Govindan博士分别负责TIRF/多光子组件和教育/培训。该设施由LCCC，退款费和LCCC癌症中心支持补助金（2 P30-CA-51008）资助。公共卫生相关性：这项建议的资金购买激光共聚焦升级到研究显微镜将主要支持研究在文森特T。隆巴迪癌症中心，指定的NIH综合癌症中心和乔治敦大学医学中心和主校区的其他研究人员。这种激光共聚焦升级将有助于识别蛋白质和亚细胞结构的细胞位置，这些蛋白质和亚细胞结构可能导致肿瘤细胞侵袭和血管生成，高血压，真菌和疟疾感染的发展以及免疫力的发展，以及有效的药物靶向。该显微镜将被放置在一个预先存在的共享资源中，由伦巴第研究人员的核心使用，并提供给其他乔治敦研究人员，这些研究人员也得到了NIH资助的赠款。

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.