Total Internal Reflection Fluorescence (TIRF) and Multidimensional Single Molecule Fluorescence (MSMM) microscopy of plant proteins

这个跨学科的项目将率先将最先进的多维单分子荧光显微镜（MSMM）应用到植物发育研究领域。利用这项技术，我们在达斯伯里的同事已经能够在正常生理条件下的活细胞中，量化表皮生长因子受体在其构成和活性状态下的寡聚化状态和构象变化。我们提出利用MSMM和贝叶斯分割分析来分析荧光标记的植物蛋白。这项技术还使用了一种最先进的图像分析算法，该算法最初是为从极其微弱、嘈杂和模糊的天文数据中提取定量信息而开发的。在单分子研究中，来自单个受体的微弱信号与活细胞荧光背景的不可避免的存在相结合，意味着数据中的基本信息可能被背景噪声模糊和/或隐藏。成像分析方法旨在实现受体数据上的预先存在的信息，这些信息可以明确地包含在图像分析算法中，从而创建更强大的算法，并证明其结果值得信赖。结合这些图像分析方法，该仪器将作为分子尺度上的精确光谱尺和量角器，提供有关天然环境中荧光标记蛋白质的构象和结合特性的数据。荧光标记的蛋白质将在洋葱表皮细胞、原生质体和细胞悬浮培养物中短暂表达，并用于TIRFM。MSMM需要极低水平的荧光，这是不太可能产生使用天然启动子或CaMV35S。因此，snap标签的使用将受到测试。该系统依赖于在感兴趣的蛋白和180个氨基酸的小蛋白SNAP-tagTM （Covalys）之间创建融合蛋白，该蛋白可以在体内用多种荧光或亲和底物进行共价标记。这样做的好处是，通过改变标记量，可以独立于表达水平产生不同水平的信号，从而产生MSMM所需的低水平荧光。SNAP-tag系统将用于生成融合蛋白，这些融合蛋白可以在体内进行共价标记，以获得MSMM所需的低水平荧光。该项目将提供机会将该技术整合到植物科学研究领域。

This interdisciplinary project will pioneer implementation of state-of-the-art multidimensional single molecule fluorescence microscopy (MSMM) into the Plant developmental Research field. Using this technique our colleagues at Daresbury have been able to quantify the oligomerisation state and conformational changes of the epidermal growth factor receptor in its constitutive and active states, in living cells under normal physiological conditions. We propose to use MSMM and Bayesian segmentation analysis to analyse plant proteins labelled with fluorescent tags. This technique also uses a state-of/the art image analysis algorithm originally developed for extracting quantitative information from extremely faint, noisy and blurred astronomical data. In single molecule research, the combination of weak signals from single receptors with the unavoidable presence of the fluorescence background from living cells implies that essential information in the data can be blurred and/or hidden by the background noise. The imaging analysis method seeks to implement, pre-existing information on the receptor data, which can be explicitly included in the image analysis algorithms, creating a more powerful algorithm with demonstrated trustworthy results. In combination with these image analysis methods this instrument will serve as an accurate spectroscopic ruler and protractor at the molecular scale to provide data on the conformation and binding characteristics of fluorescently labelled proteins in the native environment. Fluorescently labelled proteins will be transiently expressed in onion epidermal cells, protoplasts and cell suspension cultures and used for TIRFM. MSMM requires extremely low levels of fluorescence, which are unlikely to be generated using native promoters or CaMV35S. The use of SNAP-tags will therefore be tested. This system relies upon creating a fusion protein between the protein of interest and a small 180 amino-acid protein 'SNAP-tagTM (Covalys) which can be covalently labelled in vivo with a wide range of fluorescent or affinity substrates. This has the advantage that varying levels of signal can be generated independent of expression levels, by altering the amount of labelling, which allows the generation of low-levels of fluorescence needed for MSMM. The SNAP-tag system will be used to generate fusion proteins that can be covalently labelled in vivo for low-levels of fluorescence required for MSMM. This project will provide the opportunity to integrate this technology into the Plant Sciences Research field.