Novel Lambda FRET Spectral Confocal Microscopy Imaging Method
Novel Lambda FRET Spectral Confocal Microscopy Imaging Method
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DOI：
10.1002/jemt.20633
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发表时间：
2009-01-01
影响因子：
2.5
通讯作者：
Montoya, Maria C.
中科院分区：
文献类型：
--
作者：
Megias, Diego;Marrero, Raquel;Montoya, Maria C.
我们报告了一种高度特异性、灵敏且稳健的方法，用于分析基于光谱激光扫描共焦显微镜成像的荧光共振能量转移（FRET）。 lambda FRET (lambda FRET) 算法包括在多个发射波长下对 FRET 样品进行成像，从而呈现 FRET 光谱，该光谱被分为其供体和受体分量，以获得基于像素的 FRET 效率计算。该方法使用一种新颖的离线预校准程序，基于参考反射图像的采集来进行光谱渗透校正，从而简化了方法并减少了变异性。 lambda FRET 方法使用具有可变接头长度和为此目的设计的化学计量的结构特征 FRET 标准进行了验证。在特异性、重现性和对距离变化的敏感性方面，FRET 比其他成熟的方法（例如受体光漂白和基于敏化发射的方法）表现更好。此外，λ FRET 分析不受高荧光染料光谱重叠和细胞自发荧光的影响。 lambda FRET 方法在固定细胞和活细胞成像研究中的分子内和分子间 FRET 分析中表现出出色的性能。显微镜。资源。技术。 72:1-11, 2009。(C) 2008 Wiley-Liss, Inc.
We report a highly specific, sensitive, and robust method for analyzing fluorescence resonance energy transfer (FRET) based on spectral laser scanning confocal microscopy imaging. The lambda FRET (lambda FRET) algorithm comprises imaging of a FRET sample at multiple emission wavelengths rendering a FRET spectrum, which is separated into its donor and acceptor components to obtain a pixel-based calculation of FRET efficiency. The method uses a novel off-line precalibration procedure for spectral bleed-through correction based on the acquisition of reference reflection images, which simplifies the method and reduces variability. lambda FRET method was validated using structurally characterized FRET standards with variable linker lengths and stoichiometries designed for this purpose.,FRET performed better than other well-established methods, such as acceptor photobleaching and sensitized emission-based methods, in terms of specificity, reproducibility, and sensitivity to distance variations. Moreover, lambda FRET analysis was unaffected by high fluorochrome spectral overlap and cellular autofluorescence. The lambda FRET method demonstrated outstanding performance in intra-and intermolecular FRET analysis in both fixed and live cell imaging studies. Microsc. Res. Tech. 72:1-11, 2009. (C) 2008 Wiley-Liss, Inc.