Visible and near infrared fluorescence spectral flow cytometry
Visible and near infrared fluorescence spectral flow cytometry
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DOI：
10.1002/cyto.a.22241
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发表时间：
2013-03-01
期刊：
影响因子：
3.7
通讯作者：
Novo, David
中科院分区：
文献类型：
--
作者：
Nolan, John P.;Condello, Danilo;Novo, David
在流式细胞术中测量来自单个细胞的完整发射光谱存在长期的兴趣。我们开发了流式细胞仪仪器和分析方法，使其能够常规和稳健地完成。我们的光谱流式细胞仪使用全息光栅将来自单细胞的光分散到CCD上，以进行高速波长分辨检测。定制软件允许显示和分析单细胞光谱数据，以产生新的光谱衍生参数。我们表明，熟悉的参考和校准珠可以用来定量评估仪器的性能。我们使用六种不同的量子点染色的微球比较虚拟带通滤波器的方法与经典的最小二乘（CLS）光谱解混，然后使用抗体捕获珠和CLS解混，以证明外周血单核细胞的免疫表型，使用光谱流式细胞术。最后，我们表征和评估几个近红外（NIR）发射荧光团用于光谱流式细胞术。光谱流式细胞术为单细胞分析提供了许多有吸引力的功能，包括简化的光路，高光谱分辨率和简化的多参数定量测量方法。强大的仪器，软件和分析方法的可用性将促进光谱流式细胞术应用的发展。(c)2012年国际细胞计数促进学会
There is a long standing interest in measuring complete emission spectra from individual cells in flow cytometry. We have developed flow cytometry instruments and analysis approaches to enable this to be done routinely and robustly. Our spectral flow cytometers use a holographic grating to disperse light from single cells onto a CCD for high speed, wavelength-resolved detection. Customized software allows the single cell spectral data to be displayed and analyzed to produce new spectra-derived parameters. We show that familiar reference and calibration beads can be employed to quantitatively assess instrument performance. We use microspheres stained with six different quantum dots to compare a virtual bandpass filter approach with classic least squares (CLS) spectral unmixing, and then use antibody capture beads and CLS unmixing to demonstrate immunophenotyping of peripheral blood mononuclear cells using spectral flow cytometry. Finally, we characterize and evaluate several near infrared (NIR) emitting fluorophores for use in spectral flow cytometry. Spectral flow cytometry offers a number of attractive features for single cell analysis, including a simplified optical path, high spectral resolution, and streamlined approaches to quantitative multiparameter measurements. The availability of robust instrumentation, software, and analysis approaches will facilitate the development of spectral flow cytometry applications. (c) 2012 International Society for Advancement of Cytometry