Developing a label-free single molecule imaging technology: transitioning from material science to biological science

在没有标签的情况下可视化单个蛋白质是研究天然蛋白质功能的重要一步。在这个应用程序中，我们建议将丹麦基础计量学（DFM）和Kad实验室（肯特大学）的技术结合起来，生成一个新的无标签成像工具。这将用于研究DNA修复作为一个模型生物系统。Kad实验室目前使用单分子荧光成像来研究原核生物的核苷酸切除DNA修复（NER）。这种方法对这些蛋白质所使用的物理搜索机制产生了相当大的新见解。为了获得更深入的了解，我们构建了一个高速暗场成像系统，可以测量从单个金纳米粒子标记的蛋白质中散射出来的光。该系统的一个主要缺点是金纳米颗粒（~40 nm）增加了尺寸，从而降低了蛋白质的扩散常数。因此，我们必须使用理论计算来解释附着的纳米粒子的预期贡献。理想情况下，没有标签的蛋白质成像将提供这些蛋白质如何定位其目标位点的准确测量。我们与专门测量材料基本特性的DFM合作。这些技术对于先进光学元件的开发（例如纳米结构光栅）至关重要。一种这样的系统可以检测从表面上排列的单个未标记分子发出的光散射（散射测量）。通过使用散射测量法，我们将省去金纳米粒子，而是直接检测单个蛋白质的散射信号。这项技术只需要很少的硬件开发；我们使用一个开放的光学系统，可以很容易地适应散射测量。这个博士项目的一个主要组成部分是傅里叶图像分析软件的应用，这是DFM正在进行开发的主题。

Visualising single proteins in the absence of a label represents a vital step towards studying native protein function. In this application we propose to combine the technologies available at Danish Fundamental Metrology (DFM) with that of the Kad lab (University of Kent) to generate a new label-free imaging tool. This will be used to study DNA repair as a model biological system.The Kad lab presently uses single molecule fluorescence imaging to study nucleotide excision DNA repair (NER) in prokaryotes. This approach has led to considerable new insights into the physical search mechanisms employed by these proteins. To enable further insights we have constructed a high-speed dark-field imaging system that measures the light scattered from individual gold nanoparticle labelled proteins. One key drawback of this system is that the gold nanoparticles (~40 nm) increase the size and thus reduce the diffusion constant of the protein. Therefore we must use theoretical calculations to account for the expected contribution of the attached nanoparticle. Ideally, imaging proteins without a label would provide an accurate measure of how these proteins locate their target sites.We have teamed up with DFM who specialise in measuring the fundamental properties of materials. Such technologies are crucial for advanced optical component development (for example nanostructured gratings). One such system detects light scatter from single unlabelled molecules ordered on a surface (scatterometry). By using scatterometry we will dispense with gold nanoparticles and instead detect the scatter signal from single proteins directly. This technology requires very little hardware development; we use an open optical system that can easily be adapted to accommodate for scatterometry. A major component in this PhD project is the application of Fourier image analysis software, which is the subject of on-going development at DFM.