An expanded 75-color panel of Pdots for spectral multiplexing

项目摘要 流式细胞术广泛用于基础和转化研究以及临床诊断测定。谱流 流式细胞术代表了通过使用全光谱信息的流式细胞术方法的革命性变化 从每个荧光团-通过收集整个光谱上的发射并使用多个激发源 以获得光谱“特征”或“指纹”。所得到的光谱特征用于 自动光谱分解比当前的补偿方法更简单和更有效。因此，在本发明中， 甚至可以同时使用具有高度重叠光谱的荧光团，简化了面板构造 并且扩大了最大板尺寸;细胞自发荧光的扣除也得到了改善。这些因素 联合收割机以允许更高的细胞亚群分辨率。此外，硬件可以被简化，因为 对于特定的一组染料，不需要过滤器组的特定构造。光谱流式细胞术具有 发展到主流，现在有几种商业光谱流系统可用（Cytek Aurora，Sony ID7000，BD Symphony A5 SE，Thermo Bigfoot）。 然而，染料的发展并没有跟上光谱流系统的发展。为 例如，Sony的ID 7000系统包括320 nm激发源，对于该激发源没有染料被特别地 开发我们和其他人已经开发出一种新的超亮荧光纳米粒子家族，称为Pdot， 其是塌陷成5-30 nm（可调尺寸）纳米颗粒的半导体聚合物。Pdot是明亮的， 这种材料是光稳定的，并且在表现出长的、可调的斯托克斯位移方面是独特的，从而能够产生大的彩色面板。 在这里，我们建议通过添加38种新颜色来将Pdot的多色面板从37扩展到75， 包括在320 nm处激发的新的一系列18个Pdot（没有先前的Pdot在320 nm处可激发）， 以及在355、405、488、561和640 nm处激发的20个新的Pdot，填充了 具有相同激发的现有Pdot，同时在发射峰之间保持至少20 nm的间隔 大多数发射峰相隔30 nm或更大。我们将展示这些Pdot在光谱分析中的实用性。 通过开发和验证用于主要细胞的深度免疫表型分析的75个参数面板的流式细胞术 人外周血中的亚群。Pdot的75色面板也将在高度多重组织中有用 成像（例如，组织病理学）。

Project Summary Flow cytometry is widely used in basic and translational research and in clinical diagnostic assays. Spectral flow cytometry represents a revolutionary change in the approach to flow cytometry by using full spectral information from each fluorophore—by collecting the emission over the full spectrum and using multiple excitation sources for each fluorophore to obtain a spectral “signature” or “fingerprint”. The resulting spectral signature is used in automated spectral unmixing that is simpler and more effective than current compensation methods. As a result, even fluorophores with highly overlapping spectra can be used simultaneously, simplifying panel construction and enlarging the maximum panel size; subtraction of cell autofluorescence is also improved. These factors combine to allow greater resolution of cell subpopulations. In addition, hardware can be simplified since a particular configuration of filter sets is not needed for a particular set of dyes. Spectral flow cytometry has progressed to the mainstream, with several commercial spectral flow systems now available (Cytek Aurora, Sony ID7000, BD Symphony A5 SE, Thermo Bigfoot). However, the development of dyes has not kept pace with the development of spectral flow systems. For example, Sony’s ID7000 system includes a 320 nm excitation source for which no dyes have been specifically developed. We and others have developed a new family of ultrabright fluorescent nanoparticles called Pdots, which are semiconducting polymers collapsed into 5–30 nm (tunable size) nanoparticles. Pdots are bright and photostable, and are unique in exhibiting a long, tunable Stokes shift, enabling the creation of a large color panel. Here we propose to expand the multi-color panel of Pdots from 37 to 75 by adding 38 new colors, including a new series of 18 Pdots excited at 320 nm (no previous Pdots have been excitable at 320 nm), as well as 20 new Pdots excited at 355, 405, 488, 561, and 640 nm, filling gaps between the emission peaks of existing Pdots with the same excitation while preserving a separation of at least 20 nm between emission peaks with most emission peaks separated by 30 nm or larger. We will demonstrate the utility of these Pdots in spectral flow cytometry by developing and validating a 75-parameter panel for deep immunophenotyping of major cell subsets in human peripheral blood. The 75-color panel of Pdots will also be useful in high multiplex tissue imaging (e.g., histopathology).