全光谱流式细胞术的优势。
Advantages of full spectrum flow cytometry.
机构信息
Bioscience Division, Los Alamos National Laboratory, P.O. Box 1663, MS M888, Los Alamos, New Mexico 87544, USA.
出版信息
J Biomed Opt. 2013 Mar;18(3):037004. doi: 10.1117/1.JBO.18.3.037004.
A charge coupled device-based flow-cytometer for the measurement of full spectra was implemented and characterized. The spectral resolution was better than 1.5 nm and the coefficient of variation for fluorescence from flow check beads was 5% or better. Both cell and bead data were analyzed by fitting to measured component spectra. Separation of flow check and align flow beads, which have similar spectra, was nearly identical whether using a spectral analysis or a scatter analysis. After mixing, cells stained with ethidium bromide or propidium iodide were measured at different timepoints. The contribution of these 12 nm separated emission spectra could be separately quantified and the kinetic process of the samples becoming homogeneous due to fluorophor dissociation and rebinding was observed. Principle component analysis was used to reduce noise and alternating least squares (ALS) was used to analyze one set of noise-reduced cell data without knowledge of the component spectra. The component spectra obtained via ALS are very similar to the measured component spectra. The contributions of ethidium bromide and propidium iodide to the individual spectra are also similar to those obtained via the spectral fitting procedure.
我们构建并评估了一种基于电荷耦合器件的流式细胞仪，该仪器可用于全光谱测量。其光谱分辨率优于 1.5nm，而对于荧光微球的变异系数则在 5%或更佳。细胞和微球的数据都通过对实测组份光谱进行拟合进行分析。无论使用光谱分析还是散射分析，对于具有相似光谱的流式微球和对齐微球的分离效果都几乎完全一致。在混合之后，我们对用溴化乙锭或碘化丙啶染色的细胞在不同时间点进行测量。可以分别对这两个相隔 12nm 的发射光谱的组份进行定量，并观察到由于荧光染料的离解和重新结合，样品变得均匀的动力学过程。主成分分析（PCA）用于降低噪声，交替最小二乘法（ALS）用于在不了解组份光谱的情况下对一组去噪的细胞数据进行分析。通过 ALS 得到的组份光谱与实测组份光谱非常相似。溴化乙锭和碘化丙啶对各个光谱的贡献也与通过光谱拟合过程得到的结果相似。
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