Advanced optical manipulation and imaging techniques for the accurate quantification of cellular interaction forces

At present no effective technique exists for accurately and reliably quantifying cellular interaction forces. An understanding of these forces is crucial to the development of effective vaccinations, skin grafts, tissue regeneration and provides a greater understanding of auto-immune diseases. We propose to develop novel techniques that combine optical trapping with optical sectioning microscopy in order to fully understand and probe cellular interactions. The optical trapping system will be designed specifically for this purpose and will allow the user to directly capture and manipulate the cells of interest without the need to introduce foreign bodies or 'handles' to the sample. The cells will be trapped with a laser beam whose profile has been designed in order to minimize cell roll and hence improve the accuracy of the technique. The position of the cell will be monitored on a nanometer scale and from here the optical force applied by the laser beam will be known and any additional external force, for example from another cell, will be quantified. This specialized optical trapping system will be combined with structured light illumination - a form of optical sectioning microscopy. An optical sectioning microscope provides high quality images with greatly improved axial resolution over conventional widefield microscopy. This enables the user to build up a three-dimensional image, with sub-cellular level resolution, of their sample of interest. As part of this proposal an optical sectioning microscope will be combined with and optical trapping system to gain as much information as possible about the cell properties and configuration whilst quantifying the cellular interaction forces. The type of optical sectioning microscope used will be structured light illumination which can operate in fluorescence and has a similar axial resolution as confocal microscopy. The main benefit of structured light illumination is that it provides rapid imaging of the sample of interest allowing the cellular interactions and information from the image to be determined and compared in real-time. In a confocal microscope, for example, a laser beam would need to be scanned over every point in the image and the signal intensity record, where as in structured light illumination a minimum of three images can be taken and then processed to produce an optically sectioned image. With the technical challenges tackled and the system perfected in order to accurately and reliably quantify cellular interaction forces, it will be employed to answer some fundamental life science questions. There are several scientists currently supporting this proposal and they are interested in the level of interactions between immune cells and how this interaction can be modified and the adhesion properties of a new hydrogel matrix for three-dimensional cell culture.

Gaining a deeper understanding of cellular interactions and developing a methodology that enables the forces involved to be quantified will, in the long term, have a potential impact on healthcare, enhancing quality of life. The applications detailed in the case for support focus on the areas of immunology and tissue regeneration but it is anticipated that, in the future, many more equally relevant applications of this technology will immerge. Quantifying the forces present in the immune system will aid development of successful vaccinations and provide a greater understanding of auto-immune diseases, such as rheumatoid arthritis. In a link between the immunologists and bioengineers interested in the outcomes of this research, there is a need to understand the impact of ions on the immune system in relation to the long term health implications of metallic hip replacements. Understanding cell adhesion forces with collagen and hydrogel matrices, will impact on the development of more effective tissue regeneration techniques. The Scientists interested in this project having a particular interest in skin and kidney tissue substitutes, to help burns victims and transplant surgery respectively. The long term healthcare impact of this proposal could have wide-ranging benefit across society as a whole. Central to the remit of the Institute of Photonics is undertaking industrially relevant research in the area of photonics. The Institute gains approximately a third of all funding from industrial collaborations, and is therefore uniquely placed to fully exploit any commercial benefits of this research. The Institute has a full-time Business Development Manager (Simon Andrews) and Technologist (Gareth Valentine), their roles dedicated to enabling collaboration between industry and academia. Simon Andrews regularly exhibits at major technical conferences, participates in business seminars and knowledge transfer events. The role of the Technologist is to work on short-term industry-funded developmental work, allowing the Institute to react quickly and effectively to the demands of industrial sector. The roles of the Business Development Manager and Technologist will be fully utilized to explore any commercial benefit of this project. There are a number of companies selling optical sectioning microscopes (e.g. Ziess, Nikon, Leica and Oylmpus) and optical trapping systems (e.g. Elliot Scientific, a UK based company, and Cell Robotics) and, where appropriate, communications with these organization will be sort throughout the project. Dr. A. Wright has experience in the promotion and communication of Science to the general public and wider community. She has been involved in specific promotion events at Glasgow Science Centre and the National Museum of Scotland, and is a member of the Rank Prize Fund Opto-Electronic College (OEC). The OEC aims to develop photonics kits and experiments that can be introduced to Secondary Schools across the UK to promote Physics and Science. The kits are currently being trialed in selected schools across Scotland. Avenues for successful on-going dissemination to the general public will be explored throughout this project - one possibility being the Lectures in Schools scheme operated by the Royal Society of Edinburgh.