Flowcore Upgrade: Amnis ImageStream Flow Cytometer

描述(由申请人提供)：这是一项收购Amny ImageStream高速成像流式细胞仪的提案，作为NIH赞助的Wistar流式细胞术设施研究的一项额外的新技术。ImageStream系统在单一平台中结合了显微镜的视觉能力、图像分析的定量能力和流式细胞术的严谨统计能力，创造了新的实验能力。此外，当传统的流式细胞仪和显微镜结果不清楚或出乎意料时，这项技术将提供一个辅助工具。与传统的流式细胞仪相比，ImageStream的优势与形态有关，它提供了许多流式细胞仪的荧光信息(尽管细胞吞吐速率低于FLOW)，但同时识别了信号的位置，从而能够研究或跟踪发生在细胞上、细胞内或细胞间的信号事件或事件的运动。此外，将一个黑点与它的一个或多个图像联系起来的能力，可以确保你认为是罕见事件的东西真的是一个细胞，而不是一块碎片。与传统荧光显微镜相比，ImageStream的优势包括能够观察悬浮中的非贴壁细胞，这一点很重要，因为迫使这些细胞粘在载玻片上并将其固定可能会对细胞产生强烈的负面影响。此外，每秒能够同时获取数十到数百个单元的明场、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，人们可以量化数千个细胞的信号，同时仍然可以获得单个细胞的数据。普通的显微镜技术只能观察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。NIH资助的研究人员建议将这项技术应用于：核转位-对大样本上信号分子核转位的每细胞定量测量；内化-通过核形态变化和凋亡标记物的表达测量细胞凋亡；分子共定位-每细胞定量测量细胞内或细胞上一个或多个分子的共同联系；免疫突触-定量鉴定与两个相互作用的细胞(通常是T细胞和抗原提呈细胞(APC))接触点招募的分子的偶联物。综上所述，这项提案中提出的仪器使用建议将促进NIH资助的癌症生物学、传染病、自身免疫和疫苗开发方面的研究。 公共卫生相关性：所要求的Amny ImageStream Analyzer仪器将使Wistar能够扩大对最先进细胞分析的访问，以支持NIH资助的多个项目，重点是转录、自身免疫、传染病、癌症生物学、基因治疗和疫苗开发的细胞分析。

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.