An expanded 75-color panel of Pdots for spectral multiplexing

项目摘要 流式细胞术被广泛应用于基础研究和转译研究以及临床诊断分析。谱流 流式细胞术代表了使用全光谱信息的流式细胞术方法的革命性变化 来自每个荧光团-通过收集全光谱的发射并使用多个激发源 对于每个荧光团，获得一个光谱“签名”或“指纹”。生成的光谱签名用于 自动光谱分解，比当前的补偿方法更简单、更有效。结果, 即使是光谱高度重叠的荧光团也可以同时使用，从而简化了面板结构 并扩大了最大面板尺寸；细胞自体荧光的减法也得到了改善。这些因素 结合起来，以允许更高的细胞亚群分辨率。此外，硬件还可以简化，因为 对于特定的染料组，不需要过滤器组的特定配置。光谱流式细胞术有 发展成为主流，现在有几个商用频谱流动系统(Cytek Aurora，Sony ID7000，BD交响乐A5 SE，Thermo BigFoot)。 然而，染料的发展并没有跟上光谱流动系统的发展步伐。为 例如，索尼的ID7000系统包括一个320 nm的激发源，没有专门为其指定染料 发展起来的。我们和其他人开发了一种新的超亮荧光纳米颗粒家族，称为Pdots， 它们是半导体聚合物，坍塌成5-30 nm(尺寸可调)纳米颗粒。点是明亮的，而且 照片稳定，独特之处在于表现出长的、可调的斯托克斯漂移，从而能够创建大型彩色面板。 在这里，我们建议通过添加38种新颜色来将Pdots的多色面板从37种扩展到75种， 包括一系列新的在320 nm激发的18个点(以前没有一个点在320 nm处是可激发的)，如 以及在355、405、488、561和640 nm激发的20个新的光点，填补了发射峰之间的空隙 具有相同激发的现有Pdots，同时保持发射峰之间至少20 nm的间隔 大多数发射峰间隔30 nm或更大。我们将演示这些点在光谱中的用途 建立和验证75个参数的主细胞深度免疫表型的流式细胞术 人类外周血中的亚群。Pdots的75色面板在高多样性组织中也将有用 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).