Imaging FRET between spectrally similar GFP molecules in single cells
Imaging FRET between spectrally similar GFP molecules in single cells
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DOI：
10.1038/84443
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发表时间：
2001-02-01
影响因子：
46.9
通讯作者：
Bastiaens, PIH
中科院分区：
文献类型：
--
作者：
Harpur, AG;Wouters, FS;Bastiaens, PIH
荧光共振能量转移(FRET)1检测由绿色荧光蛋白(GFP)2变异体组成的融合结构中的荧光共振能量转移(FRET)1，绿色荧光蛋白2变异体被酶修饰或与配体结合后改变构象，从而能够检测生理过程，如钙离子释放3、蛋白酶4、5、6、7和激酶活性8。目前的FRET显微镜技术仅限于使用光谱不同的GFP，如蓝色或青色供体与绿色或黄色受体相结合。蓝色或青色GFP具有亮度较低和自发荧光的缺点。这里提出了一种FRET成像方法，它通过利用荧光寿命成像显微镜(FLIM)9、10、11、12测定供体/受体结合发射的荧光寿命来避免对GFP的光谱分离。该技术提供了一种灵敏、可重复性和内部校准的FRET测量方法，可以用于光谱相似且明亮的黄色和绿色荧光蛋白(EYFP/EGFP)，这对以前不能用于FRET应用的荧光蛋白。我们通过监测单个细胞在凋亡过程中的caspase活性，证明了这种方法在单细胞信号分析中的好处。
Fluorescence resonance energy transfer (FRET) 1 detection in fusion constructs consisting of green fluorescent protein (GFP) 2 variants linked by a sequence that changes conformation upon modification by enzymes or binding of ligands has enabled detection of physiological processes such as Ca 2+ ion release 3, and protease 4, 5, 6, 7 and kinase activity 8. Current FRET microscopy techniques are limited to the use of spectrally distinct GFPs such as blue or cyan donors in combination with green or yellow acceptors. The blue or cyan GFPs have the disadvantages of less brightness and of autofluorescence. Here a FRET imaging method is presented that circumvents the need for spectral separation of the GFPs by determination of the fluorescence lifetime of the combined donor/acceptor emission by fluorescence lifetime imaging microscopy (FLIM) 9, 10, 11, 12. This technique gives a sensitive, reproducible, and intrinsically calibrated FRET measurement that can be used with the spectrally similar and bright yellow and green fluorescent proteins (EYFP/EGFP), a pair previously unusable for FRET applications. We demonstrate the benefits of this approach in the analysis of single-cell signaling by monitoring caspase activity in individual cells during apoptosis.