Biosensor FRET detection by the phasor approach to fluorescence lifetime imaging microscopy (FLIM)
Biosensor FRET detection by the phasor approach to fluorescence lifetime imaging microscopy (FLIM)
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发表时间：
2012
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通讯作者：
Elizabeth Hinde;M. Digman;Christopher M. Welch;Klaus M. Hahn;Enrico;Gratton
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作者：
Elizabeth Hinde;M. Digman;Christopher M. Welch;Klaus M. Hahn;Enrico;Gratton
我们在这里提出的相量方法，生物传感器FRET检测荧光寿命成像显微镜（FLIM），并表明这种方法的数据表示是强大的生物传感器设计以及固有的细胞环境的荧光文物。我们证明了这一属性的一系列双链和单链生物传感器报告的本地化的Rac 1和RhoA活性，同时进行伴随的比率FRET分析所获得的FLIM数据的广义极化（GP）的方法。然后，我们评估和比较这两种方法的能力，定量图像生物传感器FRET信号作为时间和空间的函数。我们发现，在相量图中的寿命分析，每个分子物种被转换成一个二维坐标系，其中独立的荧光团的混合物可以区分由于FRET寿命的变化。这使得能够在图像的每个像素中量化双链生物传感器的自由和结合状态或单链生物传感器的低和高FRET种类的分数贡献。每种生物传感器设计固有的物理特性也可以通过相量分析准确表征;因此，这种方法可以用于在开发阶段通知生物传感器优化。我们相信，随着生物传感器变得越来越复杂，并与其他荧光分子工具，生物传感器FRET检测的相量方法FLIM不仅将成为他们的使用，而且他们的进步势在必行。生物传感器（RBD-Citrine-1 L-ECFP-RhoA-kRas）。
We present here the phasor approach to biosensor FRET detection by fluorescence lifetime imaging microscopy (FLIM) and show that this method of data representation is robust towards biosensor design as well as the fluorescence artifacts inherent to the cellular environment. We demonstrate this property on a series of dual and single chain biosensors which report the localization of Rac1 and RhoA activity, whilst performing concomitant ratiometric FRET analysis on the acquired FLIM data by the generalized polarization (GP) approach. We then evaluate and compare the ability of these two methods to quantitatively image biosensor FRET signal as a function of time and space. We find that with lifetime analysis in the phasor plot each molecular species is transformed into a two dimensional coordinate system where independent mixtures of fluorophores can be distinguished from changes in lifetime due to FRET. This enables the fractional contribution of the free and bound state of a dual chain biosensor or the low and high FRET species of a single chain biosensor to be quantified in each pixel of an image. The physical properties intrinsic to each biosensor design are also accurately characterized by the phasor analysis; thus this method could be used to inform biosensor optimization at the developmental stage. We believe that as biosensors become more sophisticated and are multiplexed with other fluorescent molecular tools, biosensor FRET detection by the phasor approach to FLIM will not only become imperative to their use but also their advancement. biosensor(RBD-Citrine-1L-ECFP-RhoA-kRas).