Research Core (Amnis ImageStreamX Core)

我们建议建立一个新的Amnis ImageStreamX核心。显微镜可以成像细胞学重排，细胞：细胞相互作用，蛋白质和膜定位，以及细胞信号事件。然而，这样的方法既不是高通量的，也不是定量的。它们也不适于实时信号传导或易于从混合物中分离确定的细胞类型。考虑到这些限制，耶鲁大学医学院（YSM）通过共享工具获得了Amnis ImageStreamX。Amnis ImageStreamX是一种多光谱成像仪器，可采集12个通道的细胞图像。通过收集每个样本的大量图像并提供基于图像的特征的数字表示，ImageStreamX将标准显微镜的每个细胞信息内容与标准流式细胞术中大样本量提供的统计学显著性相结合。ImageStreamX的最佳应用是利用其定位和定量细胞上、细胞内或细胞间信号分布的能力。这是YSM唯一的此类仪器。虽然Amnis有重要的发现，但该仪器显然没有得到充分利用。尽管在试验期间，来自Amnis的专家协助计划实验，运行样品和现场分析数据3天，但最初的反应很热烈。在这短短的时间内，从10名独立调查员那里获得了10个项目的初步数据。尽管这一早期成功是通过现场专家操作员实现的，但Amnis持续使用的模型是作为用户操作的4色流式细胞仪处理。这种模式不能自我维持。该技术的关键方面，特别是对大量数据集的必要分析，使得Amnis的最佳使用比流式细胞仪更复杂，因此更难获得。该提案旨在通过应用P30 2P核心的经验教训来弥补这一点，其中专门和经验丰富的操作员参与工作的所有阶段，包括实验规划，用户培训，成像，数据分析的援助和培训。我们建议将相同的模型应用于羊膜，以增加其对风湿性和免疫性疾病研究的影响，以及介导其发病机制的途径。

We propose to establish a new Amnis ImageStreamX Core. Microscopy can image cytological rearrangements, cell:cell interactions, protein and membrane localization, and cell signaling events. However, such methods are neither high throughput nor quantitative. Nor are they adapted for real-time signaling or facile for the separation of defined cell types from mixtures. With these limitations in mind, Yale School of Medicine (YSM) acquired through a shared instrument grant an Amnis ImageStreamX. The Amnis ImageStreamX is a multispectral imaging instrument that acquires 12 channels of cellular imagery. By collecting large numbers of images per sample and providing numerical representation of image-based features, the ImageStreamX combines the per cell information content of standard microscopy with the statistical significance afforded by large sample sizes in standard flow cytometry. The best applications for the ImageStreamX take advantage of its abilities to locate and quantitate the distribution of signals on, within or between cells. This is the only such instrument at YSM. While there have been important discoveries made with the Amnis, the instrument is clearly underutilized. This is despite an enthusiastic initial response during a trial period wherein an expert from Amnis assisted in planning experiments, running samples and analyzing data on-site for 3 days. During this short time, preliminary data were obtained for 10 projects from 10 independent investigators. Whereas this early success was achieved with an expert on-site operator, the model applied for ongoing use of the Amnis was to treat as a user-operated 4-colorfiow cytometer. This model was not self-sustaining. Key aspects of the technology and in particular the required analysis of massive data sets rendered optimal use of the Amnis more complicated than a flow cytometer and therefore less accessible. This proposal seeks to remedy this by applying lessons learned by the P30 2P Core for which a dedicated and experienced operator participates in all phases of the work including experimental planning, training of users, imaging, assistance and training in data analysis. We propose to apply the same model to the Amnis so as to augment its impact on the study of rheumatic and immunological diseases, and the pathways that mediate their pathogenesis.