Flowcore Upgrade: Amnis ImageStream Flow Cytometer

描述（由申请人提供）：这是一份申请获得Amnis ImageStream高速成像流式细胞仪的提案，作为nih资助的Wistar流式细胞仪设施研究的额外新技术。ImageStream系统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统计严谨性结合在一个平台上，创造了新的实验能力。此外，当传统流式细胞术和显微镜检测结果不明确或意外时，该技术将提供一种辅助工具。与传统流式细胞术相比，ImageStream的优势在于形态学，它提供了流式细胞仪的大部分荧光信息（尽管细胞吞吐量比流式细胞仪低），但同时可以识别信号的位置，从而可以研究或跟踪信号事件或发生在细胞上、细胞内或细胞之间的事件的运动。此外，将一个斑点与其图像联系起来的能力使您能够确保您认为罕见的事件确实是一个细胞，而不是一块碎片。与传统的荧光显微镜相比，ImageStream的优点包括能够观察悬浮中的非贴壁细胞，这一点很重要，因为强迫这些细胞粘附在载玻片上并固定它们可能对细胞产生强烈的负面影响。此外，能够同时以每秒数十到数百个细胞获取明场、暗场和4个荧光通道图像，从而实现真正的统计有效数据分析。此外，人们可以在获得单个细胞数据的同时量化数千个细胞的信号。普通的显微镜技术只能看到一个群体中的几百个细胞，这使得罕见事件分析变得困难和不切实际，如果不是不可能的话。美国国立卫生研究院资助的研究人员建议将这项技术应用于：核易位-大样本上信号分子核易位的定量每细胞测量；内化-通过细胞核形态学变化和凋亡标志物的表达来测量细胞凋亡分子共定位。单个细胞内或细胞上一个或多个分子的共结合的定量测量。免疫突触-定量鉴定结合物与分子招募到两个相互作用的细胞，通常是T细胞和抗原呈递细胞（APC）之间的接触点。综上所述，本提案中提出的仪器使用将促进NIH资助的癌症生物学、传染病、自身免疫和疫苗开发研究。

DESCRIPTION (provided by applicant): This is a proposal to acquire an Amnis ImageStream high speed imaging flow cytometer as an additional, new technology for NIH-sponsored research in Wistar's Flow Cytometry Facility. The ImageStream system 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 new experimental capabilities. In addition this technology would provide an adjunct tool to have when traditional flow cytometry and microscopy results are either unclear or unexpected. The advantages of the ImageStream as compared to traditional flow cytometry are associated with morphology, providing much of the fluorescent information of a flow cytometer (though at a lower cell throughput rate than flow), but at the same time identifying the location of the signal, which enables the study or tracking of signaling events or the movement of events that happen either on, within or between cells. Also, the ability to associate a spot with its image or images gives the ability to be sure that what you think is a rare event is truly a cell and not a piece of debris. The advantages of the ImageStream as compared to traditional fluorescence microscopy include the ability to look at non-adherent cells in suspension, important because forcing these cells to stick to slides and immobilize them could have strong negative effects on the cells. In addition, the ability to acquire brightfield, darkfield and 4 fluorescent channel images simultaneously on tens to hundreds of cells per second allows for true statistically valid data analysis. Plus, one can quantify signal on thousands of cells while still obtaining individual cell data. Normal microscopy techniques look at only a few hundred cells in a population, making rare event analysis difficult and impractical, if not impossible to do. NIH-funded investigators propose to apply this technology towards: Nuclear Translocation - Quantitative per-cell measurement of nuclear translocation of signaling molecules on large samples; Internalization - Measurement of apoptosis by nuclear morphology change and expression of apoptotic markers; Molecular Co-localization - Quantitative per- cell measurement of the co-association of one or more molecules within or on a cell; Immune Synapse - Quantitative identification of conjugates with molecules recruited to the point of contact between two interacting cells, typically a T cell and an antigen presenting cell (APC). Taken together, the proposed instrumentation usage as justified in this proposal will advance NIH funded research in cancer biology, infectious disease, autoimmunity and vaccine development. PUBLIC HEALTH RELEVANCE: The requested Amnis ImageStream Analyzer instrument will enable Wistar to extend access of state-of-the-art cell analysis in support of multiple NIH-funded projects focused on cellular analysis of transcription, autoimmunity, infectious disease, cancer biology, gene therapy, and vaccine development.