Hyperspectral cytometry at the single-cell level using a 32-channel photodetector
Hyperspectral cytometry at the single-cell level using a 32-channel photodetector
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DOI：
10.1002/cyto.a.21120
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发表时间：
2012-01-01
期刊：
影响因子：
3.7
通讯作者：
Robinson, J. Paul
中科院分区：
文献类型：
--
作者：
Gregori, Gerald;Patsekin, Valery;Robinson, J. Paul
尽管细胞分析技术最近取得了进展，但细胞的快速分类仍然是一项非常困难的任务。在现有的技术中，流式细胞术（FCM）被认为是特别强大的，因为它能够以高流速（高达每秒数千个颗粒）对单个生物颗粒进行多参数分析。此外，FCM是非破坏性的，并且可以对活细胞进行流式细胞术分析。根据仪器的不同，FCM中同时可检测的荧光信号的电流极限约为8-15。获得多参数测量是一项非常复杂的任务，荧光光谱重叠补偿的必要性产生了许多额外的困难来解决。此外，为了获得良好分离的单个光谱带，需要非常复杂的滤光器组。本研究描述了构建基于32通道PMT阵列检测器、相位体全息光栅和快速电子板的下一代流式细胞仪的关键组件和原理。该系统能够在单细胞水平上进行全光谱数据收集和光谱分析。如使用荧光微球和用抗体混合物（CD 45/FITC、CD 4/PE、CD 8/ECD和CD 3/Cy 5）标记的淋巴细胞所证明的，所提出的技术能够在< 5 μ s内从流过激光束的单个颗粒同时收集32个窄带荧光。这32个离散值提供了每个单个颗粒（细胞）的完整荧光发射光谱的代表。然后进行高级统计分析以分离淋巴细胞的各种簇。针对每个聚类计算的平均谱已被用于表征抗体的相应组合，并且因此鉴定各种淋巴细胞亚群。该流式细胞仪强大的数据收集能力为先进的分析方法提供了重要的机会，包括光谱分离和无监督或监督分类。(C)2011年国际细胞计数促进学会
Despite recent progress in cell-analysis technology, rapid classification of cells remains a very difficult task. Among the techniques available, flow cytometry (FCM) is considered especially powerful, because it is able to perform multiparametric analyses of single biological particles at a high flow rate-up to several thousand particles per second. Moreover, FCM is nondestructive, and flow cytometric analysis can be performed on live cells. The current limit for simultaneously detectable fluorescence signals in FCM is around 8-15 depending upon the instrument. Obtaining multiparametric measurements is a very complex task, and the necessity for fluorescence spectral overlap compensation creates a number of additional difficulties to solve. Further, to obtain well-separated single spectral bands a very complex set of optical filters is required. This study describes the key components and principles involved in building a next-generation flow cytometer based on a 32-channel PMT array detector, a phase-volume holographic grating, and a fast electronic board. The system is capable of full-spectral data collection and spectral analysis at the single-cell level. As demonstrated using fluorescent microspheres and lymphocytes labeled with a cocktail of antibodies (CD45/FITC, CD4/PE, CD8/ECD, and CD3/Cy5), the presented technology is able to simultaneously collect 32 narrow bands of fluorescence from single particles flowing across the laser beam in < 5 mu s. These 32 discrete values provide a proxy of the full fluorescence emission spectrum for each single particle (cell). Advanced statistical analysis has then been performed to separate the various clusters of lymphocytes. The average spectrum computed for each cluster has been used to characterize the corresponding combination of antibodies, and thus identify the various lymphocytes subsets. The powerful data-collection capabilities of this flow cytometer open up significant opportunities for advanced analytical approaches, including spectral unmixing and unsupervised or supervised classification. (C) 2011 International Society for Advancement of Cytometry