Simultaneous measurements of fluorescence lifetimes, anisotropy, and FRAP recovery curves

Simultaneous measurements of fluorescence lifetimes, anisotropy, and FRAP recovery curves
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同时测量荧光寿命、各向异性和 FRAP 恢复曲线

DOI:
10.1117/12.875151
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发表时间:
2011
期刊:
--
影响因子:
--
通讯作者:
Levitt J
Levitt J
中科院分区:
--
文献类型:
--
作者:
Levitt J

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我们介绍了荧光寿命成像(FLIM)和荧光各向异性成像以及在单个实验中记录的绿色荧光蛋白(GFP)标记的活细胞的平移扩散测量。该实验装置允许在光漂白后荧光恢复(FRAP)系列的每一帧中测量时间和偏振分辨荧光图像。我们已经在均相溶液中使用罗丹明123验证了该方法,然后测量了标记有CDC42-GFP的活A431细胞,对于这些细胞,FRAP恢复显示出固定的部分,蛋白质的旋转移动性受到阻碍,而荧光寿命在整个细胞中相当均匀。通过消除连续测量来提取荧光寿命和分子扩散系数的需要,我们消除了在连续实验期间由于样品形态变化和过度光漂白而产生的伪影。
We present fluorescence lifetime imaging (FLIM) and fluorescence anisotropy imaging along with translational diffusion measurements of living cells labelled with green fluorescent protein (GFP) recorded in a single experiment. The experimental set-up allows for time and polarization-resolved fluorescence images to be measured in every frame of a fluorescence recovery after photobleaching (FRAP) series. We have validated the method using rhodamine 123 in homogeneous solution prior to measurements of living A431 cells labelled with cdc42-GFP, for which the FRAP recovery exhibits an immobile fraction and the rotational mobility of the protein is hindered while the fluorescence lifetime fairly homogeneous across the cell. By eliminating the need for sequential measurements to extract fluorescence lifetimes and molecular diffusion coefficients we remove artefacts arising from changes in sample morphology and excessive photobleaching during sequential experiments.
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