An expanded 75-color panel of Pdots for spectral multiplexing

用于光谱复用的扩展 75 色 Pdot 面板

• 批准号: 10761598
• 负责人: Jiangbo Yu
• 金额: $ 49.96万
• 依托单位: LAMPROGEN, INC.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10761598
• 关键词: Antibodies; Autoimmune Diseases; B-Lymphocytes; Basic Science; Biological Markers; CD8-Positive T-Lymphocytes; Cell Cycle; Cells; Cellular Assay; Clinical; Color; Communicable Diseases; Compensation; Coupling; Dendritic Cells; Development; Diagnosis; Diagnostic tests; Disease; Dyes; Energy Transfer; Ensure; Exhibits; Family; Fingerprint; Flow Cytometry; Fluorescent Dyes; Fluorescent Probes; Fluorochrome; Future; Histopathology; Human; Immune; Immune response; Immunophenotyping; Label; Lasers; Mainstreaming; Malignant Neoplasms; Measures; Metabolic; Methods; Natural Killer Cells; Nature; Optics; Pamphlets; Particle Size; Polymers; Population; Proteins; Quantum Dots; Reaction; Regulatory T-Lymphocyte; Research Personnel; Resolution; Series; Signal Transduction; Sorting; Source; System; T-Lymphocyte; Techniques; Technology; Time; Tissue imaging; Translational Research; Validation; Vertebral column; Width; antibody conjugate; cancer therapy; cell analyzer; clinical diagnostics; commercialization; design; detection platform; detector; diagnostic assay; diagnostic panel; experimental study; fluorophore; improved; instrument; monocyte; monomer; multiplexed imaging; nanoparticle; peripheral blood; personalized medicine; preservation; public health relevance; ultraviolet

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).

项目概要 流式细胞术广泛应用于基础和转化研究以及临床诊断分析。 流式细胞术利用完整的光谱信息代表了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 为每个荧光团获得光谱“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、更有效。 甚至可以同时使用具有高度重叠光谱的荧光团，从而简化面板构造 并且扩大了最大面板尺寸；并且还改进了细胞自发荧光的减法。 结合起来可以提高细胞亚群的分辨率。此外，还可以简化硬件，因为 光谱流式细胞术不需要特定的滤光片组配置。 已发展成为主流，现已推出多种商用光谱流系统（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步伐。 例如，索尼的 ID7000 系统包括一个 320 nm 激发源，尚未专门针对该激发源使用任何染料。 我们和其他人开发了一种新的超亮荧光纳米粒子家族，称为 Pdots， 它们是塌缩成 5-30 nm（可调尺寸）纳米粒子的半导体聚合物，明亮且明亮。 光稳定，并且在表现出长的、可调谐的斯托克斯位移方面是独一无二的，从而能够创建大型彩色面板。 在这里，我们建议通过添加 38 种新颜色将 Pdot 的多色面板从 37 种扩展到 75 种， 包括在 320 nm 激发的一系列新的 18 个 Pdot（之前没有 Pdot 在 320 nm 激发），如 以及 20 个在 355、405、488、561 和 640 nm 激发的新 Pdot，填补了发射峰之间的间隙 现有的 Pdot 具有相同的激发，同时保留发射峰之间至少 20 nm 的间隔 大多数发射峰间隔 30 nm 或更大，我们将展示这些 Pdot 在光谱中的实用性。 通过开发和验证用于主要细胞深度免疫表型分析的 75 参数组来进行流式细胞术 人外周血中的子集也可用于高多重组织。 成像（例如组织病理学）。