Polarized fluorescence resonance energy transfer microscopy
Polarized fluorescence resonance energy transfer microscopy
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DOI：
10.1529/biophysj.103.036194
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发表时间：
2004-10-01
影响因子：
3.4
通讯作者：
Axelrod, D
中科院分区：
文献类型：
--
作者：
Mattheyses, AL;Hoppe, AD;Axelrod, D
目前用于活细胞的荧光共振能量转移（FRET）显微术的方法涉及在单独的相机曝光中用交替的激发颜色拍摄一系列图像。在这里，我们提出了一种新的FRET方法的基础上，偏振，只需要一个相机曝光，从而提供了更好的时间分辨率的亚细胞成分之间的动态关联的可能性。偏振FRET（p-FRET）使用来自两个正交偏振源的激发波长的同时组合，以及沿着配备有适当偏振器的发射通道三图像分离器，以同时激发和收集来自自由供体、自由受体和FRET对的荧光。基于对三种物质中的每一种的纯样品预先测量的每个发射通道中的通量，可以执行未知样品的三个偏振荧光图像的解耦以计算供体、受体和FRET对的逐像素浓度。本文介绍了这种方法的理论，并通过对活细胞中青色荧光蛋白（CFP）、柠檬酸（（Cit）一种黄色荧光蛋白变体）和连接的融合蛋白（CFP-L16-Cit、CFP-L7-Cit、CFP-L54-Cit）的混合物进行测量，实验证实了其可行性。散粒噪声，受体偏振，和FRET效率的p-FRET实验结果的统计精度的影响进行了研究，通过噪声模拟程序。
Current methods for fluorescence resonance energy transfer ( FRET) microscopy of living cells involve taking a series of images with alternating excitation colors in separate camera exposures. Here we present a new FRET method based on polarization that requires only one camera exposure and thereby offers the possibility for better time resolution of dynamic associations among subcellular components. Polarized FRET (p-FRET) uses a simultaneous combination of excitation wavelengths from two orthogonally polarized sources, along with an emission channel tri-image splitter outfitted with appropriate polarizers, to concurrently excite and collect fluorescence from free donors, free acceptors, and FRET pairs. Based upon the throughput in each emission channel as premeasured on pure samples of each of the three species, decoupling of an unknown sample's three polarized fluorescence images can be performed to calculate the pixel-by-pixel concentrations of donor, acceptor, and FRET pairs. The theory of this approach is presented here, and its feasibility is experimentally confirmed by measurements on mixtures of cyan fluorescent protein (CFP), citrine ((Cit) a yellow fluorescent protein variant), and linked fusion proteins (CFP-L16-Cit, CFP-L7-Cit, CFP-L54-Cit) in living cells. The effects of shot noise, acceptor polarization, and FRET efficiency on the statistical accuracy of p-FRET experimental results are investigated by a noise-simulation program.