Bigfoot Spectral Cell Sorter

该申请是为了获得资金，以购买生物包含的ThermoFisher Bigfoot光谱分类器（7个激光器，60个检测器）。 匹兹堡大学或方圆100英里内都没有全谱细胞分选仪。作为一种独特的乐器， Bigfoot分选机结合了高参数、高内容成像（多激光、高探测器）的优势 使用现代全光谱技术（算法光谱解混）。我们正在进行的NIH资助的研究是 由于无法直接从皮特的三个光谱中转换复杂的、经过验证的面板， 分析仪到等效的光谱细胞分选仪。在光谱方法中，从发射的光的全光谱 每个细胞由多个检测器收集，然后解混以将信号分离成单独的标记。 光谱细胞术通过允许使用新的荧光剂以及荧光剂的新组合来扩展参数空间。 先前不能在常规配置的血细胞计数器中配对在一起的现有荧光剂（即，一 检测器，单色系统）。皮特的所有分拣机都是传统的配置类型。的后果 是我们大多数或所有高度复杂的免疫表型光谱面板必须按比例缩小，适应 更少的检测器，并重新验证与现有传统分拣机的兼容性，所有这些都无法捕获 频谱分析仪揭示了原始的复杂性和新颖的信息。此外，我们目前 大量使用传统的分拣机。大脚怪的一个非常吸引人的特点是，它有两个可操作的 模式，光谱和常规。因此，大脚满足了用户对高复杂度频谱的需求。 排序的需求，也有利于我们的所有用户非常忙碌核心谁有传统的补偿需求。 所要求的Bigfoot光谱分选仪将位于Pitt's的集中统一流式细胞术核心内。 医学院。核心服务超过180个PI和500个个人用户。对于拟议的文书，主要 用户包括10名Pis（9名NIH资助），另外7名Pis为次要用户。这些私家侦探加起来 NIH资助的直接费用来自至少5个不同的研究所。一个主要的研究重点是在广泛的领域， 免疫学和相关免疫疾病，与姐妹学科，如移植，皮肤病学，血管 生物学、传染病和肿瘤学也有很好的代表性。我们的初步和演示工作向我们证明， 这台仪器将真正让我们的研究人员在他们的研究中开辟新天地。大脚怪将会是 统一流式细胞术中心的工作人员精心照顾，该中心拥有广泛的基础设施和文化 支持小组设计，监督仪器操作，并确保其在财政上自给自足。 除了提供独特的和急需的光谱分选能力，该仪器将适应 对可配置分选的需求，包括6路分选到管中和直接分选到10 ×芯片中，并允许 更好的检测和更有效的分类。分拣机有一个集成的II级生物安全柜。的 要求大脚将提供第一个国家的最先进的光谱分选仪器在皮特，以满足 我们的用户群的高复杂性、高内容需求和先进的研究计划。

This request is for funds to purchase a biocontained ThermoFisher Bigfoot spectral sorter (7 laser, 60 detector). There is no full-spectrum cell sorter at the University of Pittsburgh or within 100 miles. Unique as an instrument, the Bigfoot sorter combines the strengths of high-parameter, high-content imaging (multi-laser, high-detector) with modern full-spectrum technologies (algorithmic spectral unmixing). Our ongoing NIH-funded research is hampered by the inability to directly transition complex, validated panels from Pitt's three spectral analyzers to an equivalent spectral cell sorter. In spectral approaches, the full spectrum of light emitted from each cell is collected by multiple detectors followed by unmixing to separate the signal into individual markers. Spectral cytometry expands parameter space by permitting the use of new fluors and also new combinations of existing fluors that could not previously be paired together in conventionally configured cytometers (i.e., one detector, one color systems). All of the sorters at Pitt are the conventional configuration type. The consequence is that most or all of our highly complex immunophenotypic spectral panels have to be scaled down, adapted to fewer detectors, and re-validated for compatibility with existing conventional sorters, all of which fails to capture the original complexity and novel information tantalizingly revealed by the spectral analyzers. Further, our current conventional sorters are heavily used. A very attractive feature of the Bigfoot is that it that has two operational modes, spectral and conventional. Thus, the Bigfoot meets the demands of users with high-complexity spectral sorting needs, and also benefits all users of our very busy core who have conventionally compensated needs. The requested Bigfoot spectral sorter will be housed within the centralized Unified Flow Cytometry Core in Pitt's School of Medicine. The core serves over 180 Pis and 500 individual users. For the proposed instrument, Major Users include 10 Pis (9 NIH funded) with another 7 Pis as Minor Users. Together, these Pis have over $30 Min NIH-funded direct costs from at least 5 different institutes. A major research focus is in the broad area of immunology and allied immune diseases, with sister disciplines such as transplantation, dermatology, vascular biology, infectious disease, and oncology also well represented. Our preliminary and demo work proved to us that this instrument will truly allow our investigators to break new ground in their research. The Bigfoot will be well cared for by the staff of the Unified Flow Cytometry Core, which has an extensive infrastructure and culture in place to support panel design, oversee instrument operations, and ensure that it is financially self-supporting. In addition to providing unique and much needed spectral sorting capabilities, the instrument will accommodate demands for configurable sorts including 6-way sorts into tubes and direct sorting into 1 Ox chips, and allow for better detection and efficient sorting of rarer events. The sorter has an integrated Class II biosafety cabinet. The requested Bigfoot will provide the first state-of-the-art spectral sorter instrumentation at Pitt to meet the high-complexity, high-content needs of our user base and advancing research programs.