Pdot Enabled Cyclic Imaging Cytometry

项目摘要 对于现代流式细胞术，高通量多路复用是极其重要的，因为 分析单个细胞上和细胞内大量生物分子的巨大需求。这一趋势 在精准医学和分析不断增加的免疫细胞数量的需求的推动下， 亚型（例如免疫肿瘤学）。使用流式细胞术的高度多重细胞分析， 然而，这是一个挑战。为了解决流式细胞术的这一局限性，我们建议开发一种 循环成像细胞计数（CIC）平台，可开发用于提供简单的自动化 工作流 CIC背后的概念很简单，涉及以下步骤： 用针对不同细胞标志物的荧光探针的大颜色面板检测细胞; 2）进行 灵敏和快速的荧光成像; 3）标记细胞的快速脱色; 4）迭代 步骤1-3以获得大量成像的生物标志物。虽然顺序的概念 多年来，许多研究小组已经探索了标记和脱色，特别是 在组织成像的背景下，这种方法到目前为止还不能提供相同的水平， 这是流式细胞仪常规提供的灵敏度和通量。 最近，我们和其他人开发了基于 称为Pdot的半导体聚合物采用荧光半导体的动机 将聚合物转化为纳米颗粒标记物源于许多有利的特性，例如 吸收率大，量子产率高，发射速率快，光稳定性好。的 所得Pdot显示出非常高的荧光亮度，高出102 - 104倍 比常规染料高10-103倍，取决于颗粒尺寸。 随着Pdot的发展和可用性，我们相信我们可以发展CIC，以超过 流式细胞仪提供的性能水平。 具体地，Pdot的高亮度使得能够实现高灵敏度成像和检测。 细胞生物标志物，即使是那些以非常低的表达水平存在的生物标志物， Pdot中存在的转移允许Pdot的大彩色面板的显影， 具有窄发射的明亮且可有效地脱色以用于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.