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首页> 外文会议>Optical Investigations of Cells In Vitro and In Vivo >Digital deconvolution FRET microscopy: 3D visualization of protein-protein interactions in a single living cell
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Digital deconvolution FRET microscopy: 3D visualization of protein-protein interactions in a single living cell

机译：数字解卷积FRET显微镜：单个活细胞中蛋白质-蛋白质相互作用的3D可视化

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Abstract: The spatiotemporal dynamics of dimerization of the transcription factor Pit-1 in the living cell nucleus have been visualized and monitored by expression of genetic vectors encoding green (GFP) and blue (BFP) fluorescent protein fusions and fluorescence resonance energy transfer (FRET) imaging techniques. However, in wide-field FRET imaging microscopy the FRET signals are a combination of signals from all focal planes and the fine image details are obscured by 'out-of-focus' light. Digital deconvolution FRET imaging microscopy is used here to remove the 'out-of-focus' light to improve the resolution of the protein localization in the optical axis. Cells expressing fluorescent Pit-1 fusion proteins were imaged with a high speed, high sensitivity CCD camera and a water immersion objective lens. The point spread function (PSF) of the system was used to deconvolve the donor and acceptor images which then were ratioed to obtain the FRET signal at different optical sections. These signals were used to create three-dimensional visualization of the distribution of Pit-1 protein dimers. Digitally deconvolved data with a water immersion lens has a better signal-to-noise ratio than data obtained with an oil immersion lens. The 3-D energy transfer imaging of Pit-1 protein in the nucleus of living cells offers the possibility of studying domains within the nucleus. !31
机译：摘要：通过编码绿色（GFP）和蓝色（BFP）荧光蛋白融合物以及荧光共振能量转移（FRET）的遗传载体的表达，可以观察和监测活细胞核中转录因子Pit-1二聚体的时空动态。成像技术。但是，在宽视场FRET成像显微镜中，FRET信号是来自所有焦平面的信号的组合，并且“离焦”光会掩盖精细的图像细节。此处使用数字解卷积FRET成像显微镜去除“散焦”光，以提高光轴上蛋白质定位的分辨率。用高速，高灵敏度的CCD相机和水浸物镜对表达荧光Pit-1融合蛋白的细胞成像。系统的点扩展函数（PSF）用于对供体图像和受主图像进行去卷积，然后对它们进行比例缩放，以获取不同光学部分的FRET信号。这些信号用于创建Pit-1蛋白二聚体分布的三维可视化。与使用油浸透镜获得的数据相比，使用水浸透镜进行数字解卷积的数据具有更好的信噪比。活细胞核中Pit-1蛋白的3-D能量转移成像提供了研究核内结构域的可能性。 ！31

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