Shared Instrumentation: Amnis ImageStream System

说明(由申请人提供)：申请资金用于购买Amny ImageStream多光谱成像流式细胞仪，该仪器可以对细胞群体和细胞过程进行高通量分析。该仪器本质上将显微镜的视觉能力、图像分析的定量能力和流式细胞术的严谨统计能力结合在一个平台上，创造了非凡的新实验能力。虽然注定要成为细胞分析的单一分析平台，但这项技术直到最近才出现，因此目前研究机构中的此类仪器仍然相对较少。与传统的流式细胞术相比，ImageStream还使用了来自细胞形态的数据，提供了对样本的更全面的分析，同时仍然提供了所有的荧光信息。然而，真正实现的功能是增加了通过细胞形态分析识别荧光信号在细胞内位置的能力。ImageStream还可以在细胞内或细胞间以空间/时间方式跟踪信号事件或蛋白质运动。为了确定数据中的罕见事件是真实的还是人为的，可以使用ImageStream在特定图像中可视化单个特定信号。对于门控策略的准确性来说，这也是正确的。随着扩展景深(EDF)技术的引入，ImageStream还可以生成共焦式图像。这项技术确实增强了查看鱼斑和共同定位活动的能力。由于这些技术的进步，ImageStream可以执行广泛的应用，包括：核易位、内化分子运输、分子共定位、形态分类、核焦点斑点计数和细胞凋亡检测。在这项提案中，我们提出了13项由美国国立卫生研究院资助的个人赠款，这些赠款将直接受益于获得这项新技术。具体地说，该仪器将通过应用FISH和检测磷酸化来加强基因组学研究中的项目。细胞免疫学领域的工作将大大受益于该仪器检测罕见细胞亚群的能力，以及依赖于共同定位、核易位研究、细胞凋亡、细胞间定位和形态分类的能力的癌症研究项目。ImageStream将被并入MCG癌症中心的Flow细胞仪核心设施，其日常运营将由Flow核心设施经理监督。PI是MCGCC临床和转化研究核心设施的主任，他将监督其运作，担任咨询委员会主席，并解决MCGCC管理部门出现的问题。

DESCRIPTION (provided by applicant): Funding is requested for the purchase of an Amnis ImageStream multispectral imaging flow cytometer which can perform high-throughput analysis of cell populations and cellular processes. This instrument essentially combines the visual power of microscopy, the quantitative ability of image analysis, and the statistical rigor of flow cytometry in a single platform to create exceptional new experimental capabilities. Although destined to become the single analysis platform of choice for cell analysis, this technology has only recently become available such that there are currently still relatively few of these instruments in research institutions. Compared to traditional flow cytometry, the ImageStream also uses data derived from cell morphology which provides a more comprehensive analysis of the sample, while still providing all of the fluorescent information. The feature that is truly enabling, however, is the added ability to identify the intracellular location of the fluorescence signal through cell morphological analysis. The ImageStream also makes it possible to track signaling events or protein movement in a spatial/temporal manner, within or between cells. To determine whether rare events in the data are real or due to artifacts, individual specific signals can be visualized within a specific image using the ImageStream. This also holds true for accuracy in gating strategies. With the introduction of Extended Depth of Field (EDF) technology, the ImageStream can also generate confocal-like images. This technology really enhances the ability to look at FISH spots and co-localization events. As a result of these technological advances, the ImageStream can perform an extensive range of applications including: nuclear translocation, internalization molecular trafficking, molecular co-localization, morphological classification, nuclear foci spot counting and apoptosis detection. In this proposal we present 13 individual NIH funded grants that would directly benefit from access to this novel technology. Specifically the instrument will enhance projects in genomics studies through the application of FISH and detection of phosphorylation. Work in the fields of cellular immunology will benefit considerably from the capacity of the instrument to detect rare cellular subpopulations and cancer research projects which are dependent on the ability to perform co-localization, nuclear translocation studies, apoptosis, intercellular localization and morphological classification will similarly be enhanced. The ImageStream will be incorporated into the Flow cytmoetry core facility in the MCG Cancer Center and its day to day operation will be overseen by the Flow core facility manager. The PI is the director of the MCGCC core facilities for clinical and translational research who will oversee its operation, act as chair of the advisory committee and resolve issues arising with the MCGCC administration.