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

组合光片和扫描离子电导显微镜：在活细胞中进行单分子生物学的新工具

• 批准号: EP/L027631/1
• 负责人: David Klenerman
• 金额: $ 46.79万
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2014
• 资助国家: 英国
• 起止时间: 2014 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FL027631%2F1
• 关键词: Combined; light; sheet; scanning; ion

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.

细胞有许多不同的分子，它们相互作用以响应外部信号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，以便人们可以直接观察分子在活细胞中执行其功能。该提案的目的是在可能遵循的生物过程类型方面取得重大进展。我们将利用我们开发的一种方法，将单个分子传递到细胞表面或细胞内的特定位置，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。我们的方法是基于一个直径小于100 nm的纳米移液管，将分子输送到细胞中，我们计划产生一片薄光，然后对这些分子的去向进行成像，记录它们的荧光视频。由于我们可以在传递分子时与光片打开时同步，因此我们可以重复进行相同的测量，同时最小限度地干扰细胞。我们的想法是将细胞用作“试管”来执行这些类型的实验并自动测量，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。这将用于染色细胞表面上的少量分子，触发与免疫反应相关的关键受体，然后将蛋白质α突触核蛋白的破坏性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学和生物医学问题，因为在许多情况下，一个或几个分子可以执行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。

项目成果

Three-Dimensional Super-Resolution in Eukaryotic Cells Using the Double-Helix Point Spread Function.

• DOI: 10.1016/j.bpj.2017.02.023
• 发表时间: 2017-04-11
• 期刊: Biophysical journal
• 影响因子: 3.4
• 作者: Carr AR;Ponjavic A;Basu S;McColl J;Santos AM;Davis S;Laue ED;Klenerman D;Lee SF
• 通讯作者: Lee SF

Nanoscopic Cellular Imaging: Confinement Broadens Understanding.

• DOI: 10.1021/acsnano.6b02863
• 发表时间: 2016-09
• 期刊: ACS nano
• 影响因子: 17.1
• 作者: Stephen A. Lee;A. Ponjavic;C. Siv;Steven F. Lee;J. Biteen

Three-Dimensional Super-Resolution in Eukaryotic Cells Using the Double-Helix Point Spread Function

使用双螺旋点扩散函数的真核细胞三维超分辨率

• DOI: 10.17863/cam.9529
• 发表时间: 2017
• 作者: Carr A

Weighing one protein with light.

用光称量一种蛋白质。

• DOI: 10.1126/science.aat5851
• 发表时间: 2018
• 期刊: Science (New York, N.Y.)
• 作者: Lee SF

Single-molecule visualization of DNA G-quadruplex formation in live cells.

• DOI: 10.1038/s41557-020-0506-4
• 发表时间: 2020-09
• 期刊: Nature chemistry
• 影响因子: 21.8
• 作者: Di Antonio M;Ponjavic A;Radzevičius A;Ranasinghe RT;Catalano M;Zhang X;Shen J;Needham LM;Lee SF;Klenerman D;Balasubramanian S
• 通讯作者: Balasubramanian S