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

生物系统受到许多外部因素的影响，如温度、渗透压力和力。后者发生在多种大小的尺度上，DNA修复蛋白与其他与DNA结合的蛋白发生碰撞，迁移到感染部位的免疫细胞必须推开障碍，肌肉收缩抵抗负荷。这些都是力对生物系统影响的例子。目前，我们对这些进程缺乏清楚的了解，因为没有足够的机会获得能够研究武力影响的工具。在这项提案中，我们的目标是通过在肯特大学安装名为Lumicks C-Trap的光学陷阱系统来填补这一空白。光学捕获(也称为激光镊子)是一种允许珠子或其他物体，包括囊泡、细胞或细胞器被聚焦的光束捕获的技术，其发现者去年获得了诺贝尔奖。这种类似拖拉机光束的技术使研究人员能够物理地操纵感兴趣的生物系统。这种分析的体系结构可能包括将单个DNA分子悬浮在两个珠子之间，然后通过将DNA浸入Lumicks C-Trap微流体室的不同溶液中，在DNA上组装蛋白质复合体。使用荧光来检查组装，然后可以研究系统的力依赖关系。通过在体外施加这些力，我们了解了系统的特性以及它在细胞内的自然环境中的反应，目前还不可能进行这样的测量。或者，我们可以捕获致病酵母细胞并测量它们与流室表面材料的粘附性，并且可以直接测量它们对影响粘附性的药物的反应。这个系统的用途是广泛的，在这个方案中，我们提出了七个项目，具有不同的应用范围。我们建议安装的系统具有多种组合功能，包括微流体、光学捕获、TIRF、宽场荧光和干涉反射显微镜(IRM)。后一种技术使用光的干涉来检测没有任何标签的对象。这意味着，在一些标签影响活动的实验中，使用IRM克服了这一限制。事实上，该提案中的一个项目试图使用IRM来测量膜覆盖表面中蛋白质复合体的形成。通过组合这些功能，所产生的系统非常强大，而且也是独一无二的。在英国，没有具备这种能力的系统，而全世界只有两个。我们希望确保这项技术的广泛使用，因此我们保留了25%的仪器时间供外部使用。我们将把研究人员带到肯特郡，对该系统进行培训，并在他们的生物系统上进行武力实验。肯特的环境非常适合C-陷阱，PI是使用和开发光学陷阱技术的专家，该项目还包括来自诺丁汉大学的第二位光学陷阱专家。此外，在肯特大学，我们有各种各样的调查人员，他们将接触到这一系统的能力，因此我们将实现更快的应用多样化，这反过来将把更多的调查人员带到肯特使用C-TRAP。最后，这个系统不是现有系统的附加系统，也不是我们能力的递增进步。C-TRAP真正改变了英国各地研究人员的能力，现在是支持这种工具的合适时间和合适的研究人员群体。

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.