Rare earth cryptates for the investigation of molecular interactions in vitro and in living cells
Rare earth cryptates for the investigation of molecular interactions in vitro and in living cells
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DOI：
10.1016/j.jallcom.2007.04.054
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发表时间：
2008-02-28
影响因子：
6.2
通讯作者：
Mathis, Gerard
中科院分区：
文献类型：
--
作者：
Ghose, Sraboni;Trinquet, Eric;Mathis, Gerard
我们先前开发了一种被称为均相时间分辨荧光（HTRF）的技术，该技术已成为生物测定设计中的一种参考方法。该技术利用荧光共振能量转移（FRET），这取决于作为长寿命发光供体的铕穴状化合物与匹配的荧光受体之间的紧密接近程度。通过对活细胞内的特定分子进行标记（每个分子分别用供体或受体标记）并拍摄荧光共振能量转移（FRET）的图像，将该技术扩展到体内细胞成像成为可能。时间分辨模式能够区分由紧密接近的分子产生的信号与过量未结合的荧光标记物的背景荧光以及细胞自发荧光。当使用具有较长荧光寿命的供体和具有较短寿命的受体的组合时，在FRET作用下受体的测量荧光衰减比直接激发时受体的荧光衰减更慢。这种衰减也比细胞自发荧光慢得多，并且在配备有时间选通功能相机的时间分辨荧光显微镜下，细胞自发荧光的干扰会减少。该技术在时间分辨荧光显微镜和微孔板TRF读数仪下都进行了评估。（C）2007爱思唯尔B.V.保留所有权利。
We previously developed a technique known as homogeneous time-resolved fluorescence (HTRF) which has become a reference method in the design of bioassays. The technique makes use of the fluorescence resonance energy transfer (FRET) depending of the close proximity between europium cryptate, as a long-lived luminescent donor, and a matching fluorescent acceptor. The extension of this technique to in vivo cellular imaging is possible by the labeling of specific molecules within a living cell, each labeled either with the donor or acceptor and taking images of fluorescence resonance energy transfer (FRET). The time-resolved mode allows to discriminate the signal arising from molecules in close proximity from the background fluorescence of excess amounts of unbound fluorescent marker and from cell autolluorescence. When a combination of a donor having longer fluorescence lifetime and an acceptor having shorter lifetime is used, the measured fluorescence decays of acceptors under FRET becomes slower than the acceptor fluorescence decay upon direct excitation. The decay is also much slower than cell autofluorescence, and interference of cell autofluorescence is reduced under a time-resolved fluorescence microscope with a time-gated function equipped camera. The technique was evaluated both under a time-resolved fluorescence microscope and a microplate TRF reader. (C) 2007 Elsevier B.V. All rights reserved.