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

这一跨学科项目将率先将最先进的多维单分子荧光显微镜(MSMM)应用于植物发育研究领域。使用这项技术，我们在达累斯伯里的同事已经能够量化正常生理条件下活细胞中表皮生长因子受体在其构成和活跃状态下的寡聚状态和构象变化。我们建议使用MSMM和贝叶斯分割分析来分析用荧光标记标记的植物蛋白质。这项技术还使用了最先进的图像分析算法，最初是为从极其微弱、噪声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来自活细胞的荧光背景的不可避免地存在相结合，意味着数据中的基本信息可能会被背景噪声模糊和/或隐藏。成像分析方法寻求实现关于受体数据的预先存在的信息，这些信息可以明确地包括在图像分析算法中，从而创建具有证明可信结果的更强大的算法。与这些图像分析方法相结合，该仪器将在分子水平上充当准确的光谱标尺和量角器，以提供天然环境中荧光标记蛋白质的构象和结合特征的数据。荧光标记的蛋白质将在洋葱表皮细胞、原生质体和悬浮细胞培养中瞬时表达，并用于TIRFM。MSMM需要极低水平的荧光，这是使用本地启动子或CaMV35S不太可能产生的。因此，将对SNAP标签的使用进行测试。这个系统依赖于在感兴趣的蛋白质和一个小的180个氨基酸的蛋白质SNAP-tag TM(Covalys)之间创建融合蛋白，该融合蛋白可以在体内用广泛的荧光或亲和底物共价标记。这具有的优点是，通过改变标记量，可以产生不同水平的信号，这允许产生MSMM所需的低水平的荧光。SNAP-Tag系统将被用来产生能够在体内共价标记的融合蛋白，以获得MSMM所需的低水平的荧光。该项目将为将这项技术整合到植物科学研究领域提供机会。

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.