Quantitative linear unmixing of CFP and YFP from spectral images acquired with two-photon excitation.
Quantitative linear unmixing of CFP and YFP from spectral images acquired with two-photon excitation.
复制标题
从双光子激发获取的光谱图像中定量线性分解 CFP 和 YFP。
DOI：
10.1002/cyto.a.20267
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Vogel,StevenS
中科院分区：
文献类型：
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作者：
Thaler,Christopher;Vogel,StevenS
背景光谱不同的荧光蛋白（FP）已经开发出允许活细胞中的几种蛋白质同时可视化。在大多数情况下，FP的发射光谱重叠，使得基于滤波器技术的信号分离效率低下，并且在渗漏的情况下，不准确。光谱成像可以通过称为线性解混的过程克服这些障碍。给定由多个荧光团组成的复杂光谱，线性解混可以将复杂信号减少到其单独的、加权的分量光谱。利用双光子激发的光谱成像允许收集非截断发射光谱。线性解混在这些条件下的准确性需要进行评估。MethodsCaptures包含定义的CFP和YFP的混合物被用来测试线性解混的准确性，使用双光子激发获得的光谱图像。ResultsLinear unmixing可以准确时，波长和功率匹配的参考光谱提供给算法。线性unmixing错误可能会发生由于（1）激发激光污染的发射信号，（2）FRET的存在下，（3）激发波长的选择不佳，（4）未能背景减去参考specific.ConclusionsLinear unmixing，当明智地执行，可以准确地测量CFP和YFP在混合样品中的丰度，即使当它们的相对强度范围从90：1。© 2006国际分析细胞学学会
BackgroundSpectrally distinct fluorescent proteins (FPs) have been developed permitting the visualization of several proteins simultaneously in living cells. The emission spectra of FPs, in most cases, overlap, making signal separation based on filter technology inefficient and in cases of bleed‐through, inaccurate. Spectral imaging can overcome these obstacles through a process called linear unmixing. Given a complex spectra composed of multiple fluorophores, linear unmixing can reduce the complex signal to its individual, weighted, component spectra. Spectral imaging with two‐photon excitation allows the collection of nontruncated emission spectra. The accuracy of linear unmixing under these conditions needs to be evaluated.MethodsCapillaries containing defined mixtures of CFP and YFP were used to test the accuracy of linear unmixing using spectral images obtained with two‐photon excitation.ResultsLinear unmixing can be accurate when wavelength and power‐matched reference spectra are provided to the algorithm. Linear unmixing errors can occur due to (1) excitation laser contamination of emission signals, (2) the presence of FRET, (3) poor selection of excitation wavelength, and (4) failure to background subtract reference spectra.ConclusionsLinear unmixing, when judiciously performed, can accurately measure the abundance of CFP and YFP in mixed samples, even when their relative intensities range from 90:1. © 2006 International Society for Analytical Cytology