A multi-user access laser tweezers, fluorescence and interference microscopy facility for understanding force at the molecular level

多用户访问激光镊子、荧光和干涉显微镜设备，用于了解分子水平的力

基本信息

批准号：
BB/T017767/1
负责人：
Neil Kad
金额：
$ 96.76万
依托单位：
University of Kent
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2020
资助国家：
英国
起止时间：
2020 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FT017767%2F1
关键词：
multi user access laser tweezers

项目摘要

Biological systems are affected by numerous external factors such as temperature, osmotic stress, and force. The latter occurs on multiple size scales, DNA repair proteins are subject to collisions with other proteins bound to DNA, immune cells migrating to a site of infection must push past obstacles and muscles contract against load. These are all examples of the impact of force on biological systems. At present, we lack a clear understanding of these processes because of insufficient access to instruments capable of studying the effects of force. In this proposal, we aim to fill this gap by installing an optical trapping system at the University of Kent known as the Lumicks C-trap. Optical trapping (also known as laser tweezers), the discoverers of which were awarded the Nobel prize last year, is a technique that allows beads or other objects, including vesicles, cells or organelles to be captured by a focused beam of light. This 'tractor beam'-like technology then enables investigators to physically manipulate the biological system of interest. The architecture of such assays could include suspending a single molecule of DNA between two beads and then assembling protein complexes on the DNA by dipping the DNA into different solutions made possible by the Lumicks C-trap microfluidic chamber. Using fluorescence to check for assembly the force-dependence of the system can then be investigated. By applying these forces in vitro we learn the properties of the system and how it would respond in its native environment in the cell, where it is not presently possible to perform such measurements.Alternatively, we can capture pathogenic yeast cells and measure their adhesion to materials on the flow chamber surface, and their response to drugs affecting adhesion can be directly measured. The uses of this system are vast and in this proposal we present seven projects with a diverse spectrum of applications.The system we are proposing to install possesses multiple combined functionalities, microfluidics, optical trapping, TIRF, widefield fluorescence and interference reflection microscopy (IRM). This latter technology uses the interference of light to detect objects without any label. This means that in some experiments where labelling affects activity, using IRM overcomes this limitation. Indeed, one project in this proposal seeks to use IRM to measure the formation of protein complexes in a membrane coated surface. By combining these capabilities the resulting system is very powerful, and also unique. In the UK there are no systems with this capability, and across the world there are only two. We want to ensure wide access to this technology and therefore we are reserving 25% of instrument time for external use. We will bring investigators to Kent to train on the system and to try out force experiments on their biological systems. The environment at Kent is ideal for the C-trap, the PI is an expert in the use and development of optical trapping technologies and this project also includes a second expert in optical trapping from the University of Nottingham. Also, at the University of Kent we have a diverse range of investigators that will be exposed to the capabilities of this system and therefore we will achieve more rapid diversification of application, which in turn will bring more investigators to Kent to use the C-trap. Finally, this system is not an add-on to an existing system, nor is it an incremental advance in our capabilities. The C-trap offers a genuine step change in the capabilities of researchers across the UK, and this is the right time and right group of investigators to support such an instrument.

生物系统受到许多外部因素的影响，例如温度，渗透应激和力。后者发生在多个尺寸的尺度上，DNA修复蛋白与与DNA结合的其他蛋白质发生碰撞，免疫细胞迁移到感染部位必须将障碍物越过障碍物，并且肌肉收缩降低了负载。这些都是力对生物系统的影响的例子。目前，由于无法使用能够研究武力影响的工具，我们对这些过程缺乏清晰的了解。在此提案中，我们的目标是通过在肯特大学（Lumicks C-Trap）安装光学诱捕系统来填补这一空白。光学诱捕（也称为激光镊子）去年被授予诺贝尔奖的发现者，是一种允许珠子或其他物体（包括囊泡，细胞或细胞器）的技术，可以被聚焦的光束捕获。然后，这种“拖拉机光束”技术使研究人员能够物理操纵感兴趣的生物系统。这种测定的结构可能包括将单个DNA的单个DNA悬浮在两个珠之间，然后通过将DNA浸入Lumicks C-Trap微流体腔室使DNA上的蛋白质复合物在DNA上悬浮。然后可以使用荧光检查组装系统的力依赖性。通过在体外施加这些力，我们了解系统的特性以及它在细胞中的天然环境中如何响应，目前无法执行此类测量。可以直接测量流量室表面上的材料及其对影响粘附药物的药物的反应。该系统的用途是广泛的，在该提案中，我们提出了七个具有各种应用程序的项目。我们建议安装的系统具有多种合并功能，微流体，光学诱捕，TIRF，广场荧光和干扰反射显微镜（IRM）（IRM）。后一种技术利用光的干扰来检测物体而无需任何标签。这意味着在某些实验中，标记会影响活动，使用IRM克服了此限制。实际上，该提案中的一个项目试图使用IRM来测量膜涂层表面中蛋白质复合物的形成。通过结合这些功能，最终的系统非常强大，并且也很独特。在英国，没有这种能力的系统，在世界范围内只有两个系统。我们希望确保广泛使用这项技术，因此我们保留了25％的仪器时间用于外部使用。我们将将调查人员带到肯特，以对系统进行训练，并在其生物系统上尝试实力实验。肯特的环境非常适合C-TRAP，PI是光学诱捕技术的使用和开发专家，该项目还包括来自诺丁汉大学光学诱捕的第二位专家。此外，在肯特大学，我们有各种各样的调查人员，这些调查人员将暴露于该系统的功能，因此我们将实现更快的应用程序多元化，这反过来又将为肯特带来更多的调查员来使用C-Trap 。最后，该系统不是现有系统的附加组件，也不是我们功能的增量进步。 C-trap在英国研究人员的能力方面提供了真正的步骤，这是支持这种工具的合适时间和正确的研究人员。