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

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.

Force is relevant to many biological systems, however, in many cases it remains unstudied because of the difficulties in making measurements. In this proposal, we aim to install a Lumicks optical trap (C-trap) in the School of Biosciences at the University of Kent. This system is user-friendly with simple, approachable, and largely automated software. It combines optical trapping with TIRF/widefield fluorescence imaging, microfluidics and interference reflection microscopy. Optical trapping enables physical manipulation of biological samples, from glass beads to cells - enabling forces to be applied and/or measured at the molecules to cells scale. The seven projects in this proposal all utilize different aspects of the capabilities of this system. Project 1 will use optical trapping and widefield fluorescence, 2 will use microfluidics and trapping force measurement, 3 will use IRM and trapping, 4 uses TIRF and trapping, 5 uses force feedback trapping and fluorescence, 6 uses multi-point trapping, force and fluorescence and project 7 will use IRM, trapping and microfluidics.
Only one other C-trap is present in the UK, however, that instrument does not possess this capability and is not open access. By contrast, we will dedicate 25% of instrument time to external access, facilitated by Lumicks and the Central Laser Facility (Harwell) who will bring users to the instrument for training and data collection. The longer term provision of this system has been secured by a University commitment to fund two extra years of service contract and provide a technician.
Locating the system at Kent is ideal given the optical trapping expertise of the PI, and the diverse applications Kent has to offer. However, the range of support letters and co-PIs from other institutions, demonstrate the wide appeal of this system.
It is important to note that the microscope is not an incremental advance in our capabilities, but instead is a major step forward for the whole of UK science.

Who will form our user pool? The principle beneficiaries of this investment in an optical trapping microscope will be significant portions of the academic community who aim to understand the role of force in biological systems. These investigators are currently unable to obtain access to an instrument capable of providing a correlated trapping and imaging approach, which is what the C-trap will provide. For the projects in this proposal we have identified relevance to the following BBSRC strategic priority areas: new strategic approaches to industrial biotechnology, healthy ageing across the lifecourse, combatting antimicrobial resistance and technology development for the biosciences. In addition, the projects in this proposal have relevance to the following areas of the BBSRC delivery plan: transformative technologies, understanding the rules of life, bioscience for renewable resources and clean growth, bioscience for an integrated understanding of health, enhancing people and talent, and infrastructure.
Since we are proposing to install such a versatile instrument and we will be offering access time to external investigators, it is not possible to clearly define all of the specific areas of impact this will have. Instead, it would be more relevant to consider this instrument to have potential impact across all sectors of the biosciences, and beyond. The latter interdisciplinary impact of the system is important because making advances in non-biological areas could spark new interdisciplinary projects, bringing benefits back to the biosciences.
We anticipate that the installation of a C-trap in Kent will be transformative for opportunities to perform cutting-edge force-based science in the UK. In the first instance we anticipate users of this technology to derive from the bioscience and physical science communities. Over the longer term this instrumentation will find application in areas such as material science, where force is an integral aspect of the discipline. This will likely then attract companies involved in the manufacture of active materials; therefore, we expect the impact of this instrument to be substantial. To ensure that the wider public is aware of our work we will publicise our findings through each University's Press Offices looking for publication in local and national technical and non-technical journals.
The staff training potential of this research is excellent, because we will be able to train numerous people in our annual workshops (see pathways to impact) and ad-hoc training sessions when using the instrument. There are no other instruments in the UK that offer external access and such training opportunities, therefore we will be creating a new generation of scientists and technicians with a greatly enhanced skillset. Also, we will create a user community platform as part of our web presence.
In this proposal we are seeking to understand a how forces affect biology. Installation of a Lumicks C-trap enables this, and more. Investment in this research will enable the UK to remain internationally competitive as a knowledge economy. Presently, these instruments are being installed around the world particularly in mainland Europe, Asia and the USA. It is important that the UK also invests in this area.