Fluorescence resonance energy transfer from cyan to yellow fluorescent protein detected by acceptor photobleaching using confocal microscopy and a single laser
Fluorescence resonance energy transfer from cyan to yellow fluorescent protein detected by acceptor photobleaching using confocal microscopy and a single laser
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DOI：
10.1046/j.1365-2818.2003.01100.x
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发表时间：
2003-01-01
影响因子：
2
通讯作者：
McNally, JG
中科院分区：
文献类型：
--
作者：
Karpova, TS;Baumann, CT;McNally, JG
荧光共振能量转移（FRET）的一种表现是在光漂白受体后供体荧光增加。已发表的FRET受体光漂白方法主要使用宽视场显微镜。激光扫描共聚焦显微镜可以在视野内更快、更有针对性地进行漂白，从而提高速度和准确性。在这里，我们展示了CFP和YFP的方法，这是目前可用于FRET的最通用的荧光标记。CFP/YFP FRET成像已经完成了单激光（氩），可在几乎所有的激光扫描共聚焦显微镜。在此基础上，我们还描述了利用458和514氩谱线对CFP和YFP进行双重成像的条件。我们在CFP/YFP融合中检测到FRET，也在已知的同源和异源三聚体的信号分子（tnf受体相关因子或TRAFs）之间检测到FRET。重要的是，我们证明了适当的控制是必不可少的，以避免假阳性FRET受体光漂白。我们使用两种类型的阴性对照：(a)内部阴性对照（细胞的非漂白区域）和(b)在没有受体（CFP）的情况下具有供体的细胞。我们发现，这两种类型的负控制可以产生假FRET。给定这个假FRET背景，我们描述了一种方法来区分真阳性信号。总之，我们广泛地描述了一种适用于大多数激光扫描共聚焦显微镜的简单方法，并证明了它在几个CFP/YFP伙伴之间检测FRET的可行性。
One manifestation of fluorescence resonance energy transfer (FRET) is an increase in donor fluorescence after photobleaching the acceptor. Published acceptor-photobleaching methods for FRET have mainly used wide-field microscopy. A laser scanning confocal microscope enables faster and targeted bleaching within the field of view, thereby improving speed and accuracy. Here we demonstrate the approach with CFP and YFP, the most versatile fluorescent markers now available for FRET. CFP/YFP FRET imaging has been accomplished with a single laser (argon) available on virtually all laser-scanning confocal microscopes. Accordingly, we also describe the conditions that we developed for dual imaging of CFP and YFP with the 458 and 514 argon lines. We detect FRET in a CFP/YFP fusion and also between signalling molecules (TNF-Receptor-Associated-Factors or TRAFs) that are known to homo- and heterotrimerize. Importantly, we demonstrate that appropriate controls are essential to avoid false positives in FRET by acceptor photobleaching. We use two types of negative control: (a) an internal negative control (non-bleached areas of the cell) and (b) cells with donor in the absence of the acceptor (CFP only). We find that both types of negative control can yield false FRET. Given this false FRET background, we describe a method for distinguishing true positive signals. In summary, we extensively characterize a simple approach to FRET that should be adaptable to most laser-scanning confocal microscopes, and demonstrate its feasibility for detecting FRET between several CFP/YFP partners.