Combined light sheet and scanning ion conductance microscopy : a new tool to perform single molecule biology in live cells

细胞有许多不同的分子，它们相互作用，对外部信号做出反应，并执行生命所必需的功能，在许多疾病中，这些过程会出错。最近开发的技术允许人们跟踪单个分子，这样人们就可以直接观察分子在活细胞中的功能。这项提议的目的是在可能遵循的生物过程类型上取得重大进展。我们将利用我们开发的一种方法，将单个分子传递(向下)到细胞表面或细胞内的指定位置，跟踪该分子的空间位置，然后跟踪细胞行为随时间的任何后续变化。我们的方法是基于直径小于100 nm的纳米管将分子输送到细胞，我们计划产生一张薄薄的光，然后成像这些分子的去向，记录它们的荧光视频。由于我们可以在交付分子时与打开光片时同步，所以我们可以在对细胞的干扰最小的情况下重复进行相同的测量。这个想法是将细胞作为一种“试管”来进行这些类型的实验，并使测量自动化，这样我们就可以研究许多细胞。该项目将首先调整我们现有的仪器，建立光片照明，然后编写软件来控制测量。然后，我们将进行一系列概念验证实验，以优化测量仪器和方法。这将被用来对细胞表面的少量分子进行染色，触发与免疫反应相关的关键受体，然后将具有破坏性的阿尔法突触核蛋白聚集体添加到细胞中。这种新方法应该适用于广泛的重要生物和生物医学问题，因为在许多情况下，一个或几个分子可以发挥关键功能或导致疾病。还有许多发生在哺乳动物细胞中的基本生物学过程，例如DNA的复制和修复，然后可以在未来的工作中使用这种新方法进行研究。

A cell has many different molecules that interact which each other to respond to external signals and carry out functions that are essential for life and in many diseases these processes go awry. Techniques have recently been developed that allow one to follow individual molecules, so that one can directly watch molecules perform their function in live cells. The aim of this proposal is to make a major advance in the types of biological processes that it is possible to follow. We will exploit a method that we have developed to deliver (down to) a single molecule to a defined location on the cell surface or within a cell, track the spatial position of that molecule and then any subsequent changes in the cell behaviour with time. Our method is based on a nanopipette, which is less than 100 nm in diameter, to deliver the molecules to the cell and we plan to generate a thin sheet of light to then image where these molecules go, recording a video of their fluorescence. Since we can synchronise when we deliver the molecules with when the light sheet is turned on, we can make the same measurements repetitively while minimally perturbing the cell. The idea is to use the cell as a 'test tube' to perform these types of experiments and automate the measurements so we can study many cells. The project will start by adapting our existing instrument, building the light sheet illumination and then writing software to control the measurements. We will then perform a series of proof-of-concept experiments to optimise the instrument and method of making measurements. This will be used to stain a small number of molecules on the cell surface, trigger a key receptor associated with the immune response and then add damaging aggregates of the protein alpha synuclein into cells. This new method should then be applicable to a wide range of important biological and biomedical problems since in many cases one or a few molecules can perform key functions or cause disease. There are also many fundamental biological processes that take place in mammalian cells such as the copying and repairing of DNA that could then be studied using this new method in future work.