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).

项目摘要 流式细胞仪广泛用于基础研究和翻译研究以及临床诊断测定中。光谱流 细胞仪代表通过使用全光谱信息的流式细胞仪的革命性变化 从每个荧光团中 - 通过在整个光谱上收集发射并使用多个令人兴奋的来源 让每个荧光团获得光谱“签名”或“指纹”。所得的光谱签名用于 比当前补偿方法更简单，更有效的自动频谱脉冲。因此， 即使是具有高度重叠光谱的荧光团也可以简单地使用，简化了面板的结构 并增加面板尺寸；细胞自动荧光的减法也得到了改善。这些因素 合并以更大的细胞亚群解决。此外，可以简化硬件，因为 对于特定的染料，不需要特定的过滤器集合。频谱流式细胞术具有 发展为主流，现在有几个商业光谱流系统（Cytek Aurora，Sony） ID7000，BD Symphony A5 SE，Thermo Bigfoot）。 但是，染料的发展尚未与光谱流系统的发展保持同步。为了 例如，索尼的ID7000系统包括一个320 nm的兴奋源，没有染料专门为 发达。我们和其他人开发了一个新的Ultrabright荧光纳米颗粒，称为pdots， 将半导体聚合物塌陷成5–30 nm（可调尺寸）纳米颗粒。 pdot是明亮的 光稳定性，并且在暴露长长的stokes移位方面是独一无二的，从而可以创建大型颜色面板。 在这里，我们建议通过添加38种新颜色，将多色PDOT的多色面板从37扩展到75 包括320 nm激发的新系列18个PDOT（以前没有PDOT在320 nm处令人兴奋），AS 在355、405、488、561和640 nm时激发了20个新的PDOT，填补了在发射峰之间的空白 现有的PDOT具有相同的兴奋性，同时保持在排放峰之间至少20 nm的分离 大多数发射峰分别为30 nm或更大。我们将在光谱中演示这些pdot的实用性 流式细胞仪通过开发和验证75参数面板，用于对主要细胞的深度免疫表型 人类外周血中的子集。 75颜色面板的PDOT也将在高多重组织中有用 成像（例如组织病理学）。