Maximizing the quantitative accuracy and reproducibility of Forster resonance energy transfer measurement for screening by high throughput widefield microscopy
Maximizing the quantitative accuracy and reproducibility of Forster resonance energy transfer measurement for screening by high throughput widefield microscopy
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DOI：
10.1016/j.ymeth.2013.07.040
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发表时间：
2014-03-15
期刊：
影响因子：
4.8
通讯作者：
Schaufele, Fred
中科院分区：
文献类型：
--
作者：
Schaufele, Fred
荧光蛋白 (FP) 之间的福斯特共振能量转移 (FRET) 提供了对 FP 的邻近性和方向的深入了解，作为生化相互作用的替代物以及 FP 基因融合的因子的结构。尽管 FRET 方法非常强大，但技术问题阻碍了其在生物科学中的广泛采用。准确且可重复的 FRET 显微镜测量的一个障碍源于视场内和不同视场之间的可变荧光背景。这些变化会给荧光水平的精确定量带来误差，FRET 测量的定量精度高度依赖于荧光水平。这种测量误差对于筛选活动来说尤其成问题，因为需要最小的孔间差异才能忠实地识别具有改变值的孔。高内涵筛选还取决于最大化成像的细胞数量，这最好通过低放大倍率高通量显微镜来实现。但是，低放大倍数会引入平场校正问题，从而降低背景校正的准确性，从而导致 FRET 测量的再现性较差。对于活细胞成像，常用于 FRET 分析的 FP 荧光收集通道中细胞培养基的荧光是背景误差的重要来源。这些信噪比问题因希望以具有生物学意义的水平表达蛋白质而变得更加复杂，而该水平可能仅略高于强荧光背景。这里提出了校正背景波动的技术。即使对于非平坦背景比信号高 10 倍的图像，也可以实现 FRET 的精确计算。 (C) 2013 Elsevier Inc. 保留所有权利。
Forster resonance energy transfer (FRET) between fluorescent proteins (FPs) provides insights into the proximities and orientations of FPs as surrogates of the biochemical interactions and structures of the factors to which the FPs are genetically fused. As powerful as FRET methods are, technical issues have impeded their broad adoption in the biologic sciences. One hurdle to accurate and reproducible FRET microscopy measurement stems from variable fluorescence backgrounds both within a field and between different fields. Those variations introduce errors into the precise quantification of fluorescence levels on which the quantitative accuracy of FRET measurement is highly dependent. This measurement error is particularly problematic for screening campaigns since minimal well-to-well variation is necessary to faithfully identify wells with altered values. High content screening depends also upon maximizing the numbers of cells imaged, which is best achieved by low magnification high throughput microscopy. But, low magnification introduces flat-field correction issues that degrade the accuracy of background correction to cause poor reproducibility in FRET measurement. For live cell imaging, fluorescence of cell culture media in the fluorescence collection channels for the FPs commonly used for FRET analysis is a high source of background error. These signal-to-noise problems are compounded by the desire to express proteins at biologically meaningful levels that may only be marginally above the strong fluorescence background. Here, techniques are presented that correct for background fluctuations. Accurate calculation of FRET is realized even from images in which a non-flat background is 10-fold higher than the signal. (C) 2013 Elsevier Inc. All rights reserved.