Pdot Enabled Cyclic Imaging Cytometry

项目摘要 对于现代流式细胞仪来说，高通量多路传输是极其重要的，因为 在单个细胞上和单个细胞中分析大量生物分子的巨大需要。这一趋势是 在精准医学和分析不断增加的免疫细胞数量的需要的推动下 亚型(如免疫肿瘤学)。使用流式细胞术进行高度多元化的细胞分析， 然而，这是具有挑战性的。为了解决流式细胞术的这一局限性，我们建议开发一种 循环成像细胞术(CIC)平台，可以开发提供简单的自动化 工作流程。 CIC背后的概念很简单，涉及以下步骤：1)快速标记 带有针对不同细胞标志物的大型荧光探针的彩色面板的细胞；2)执行 灵敏快速的荧光成像；3)标记细胞的快速脱染；4)迭代 步骤1-3以实现大量的成像生物标志物。虽然序贯的概念 多年来，许多研究小组一直在探索标记和脱染，特别是 在组织成像方面，这种方法到目前为止还不能提供相同的水平 通常由流式细胞仪提供的灵敏度和吞吐量。 最近，我们和其他人开发了基于以下物质的荧光纳米颗粒 被称为Pdots的半导体聚合物。采用荧光半导体的动机 聚合物制成纳米颗粒标签源于许多有利的特性，例如 吸收系数大，量子产额高，发射速率快，光稳定性好。这个 生成的Pdots显示出极高的荧光亮度，高出102-104倍 比传统染料高出10-103倍，根据颗粒大小比Qdots高10-103倍。 随着PDOTS的发展和可用，我们相信我们可以发展CIC，使其超过 由流式细胞仪提供的性能水平。 具体地说，Pdots的高亮度使高灵敏度成像和检测 细胞生物标记物，即使是那些表达水平非常低的，以及放大的能量 Pdots中存在的转印允许显影Pdots的大型彩色面板，该面板既是 光亮，发射窄，可有效脱色，用于CIC。

Project Summary For modern flow cytometry, high-throughput multiplexing is extremely important because of the great need in analyzing a large number of biomolecules on and in a single cell. This trend is driven by precision medicine and the need to analyze an ever increasing number of immune-cell subtypes (e.g. in immune-oncology). Highly multiplexed cellular analysis using flow cytometry, however, is challenging. To address this limitation of flow cytometry, we propose to develop a Cyclic Imaging Cytometry (CIC) platform that can be developed to offer a simple automated workflow. The concept behind CIC is straightforward, involving the following steps: 1) Rapid labeling of cells with a large color panel of fluorescent probes against different cellular markers; 2) Perform sensitive and rapid fluorescence imaging; 3) Rapid de-staining of the labeled cells; 4) Iterate steps 1-3 to achieve a large number of imaged biomarkers. While the concept of sequential labeling and de-staining has been explored by many research groups over the years, especially in the context of tissue imaging, this approach thus far has not been able to offer the same level of sensitivity and throughput that is routinely provided by flow cytometry. Recently, we and others have developed fluorescent nanoparticles based on semiconducting polymers called Pdots. The motivation of adapting fluorescent semiconducting polymers into nanoparticle labels stems from a number of favorable characteristics, such as large absorptivity, high quantum yield, fast emission rates, and excellent photostability. The resulting Pdots exhibit extraordinarily high fluorescence brightness, a factor of 102 - 104 higher than conventional dyes, and a factor of 10-103 higher than Qdots depending on the particle size. With the development and availability of Pdots, we believe we can develop CIC to exceed the level of performance offered by flow cytometry. Specifically, the high brightness of Pdots enables high sensitivity imaging and detection of cellular biomarkers, even those present at very low expression levels, and the amplified energy transfer present in Pdots allows the development of a large color panel of Pdots that are both bright with narrow emissions and which can be de-stained efficiently for use in CIC.