Multimodal coherent Raman and two-photon fluorescence imaging as a discovery tool in bioscience

Bioimaging is a fundamental technology underpinning bioscience and has been instrumental in unravelling the complexities of biological systems that control tissue development, differentiation and that drive disease processes. Our approach has been to combine label-free coherent Raman imaging modalities, which generate image contrast using the Raman active vibrational frequency of a given chemical, with two-photon fluorescence imaging modalities to provide insight into both subcellular and tissue organisation. Recent advances in biorthogonal chemistry which allows Raman tagging and subsequent visualisation of small drug molecules, drug delivery vehicles and cellular constituents has extended the benefit of this technology to a much wider scientific base.

Several advances in instrument design have been made since we set up our current multimodal imaging platform in 2011 which means that we can significantly enhance and extend the imaging capabilities by increasing the stability, sensitivity, accuracy and speed of our measurements, while also enabling imaging of far-red fluorophores which are becoming increasingly popular in the design of in vivo probes due to the enhanced penetration of higher wavelengths into tissue. The new set-up will provide a unique platform to carry out intravital imaging in live animals, including mice and zebrafish, providing unrivalled insight into spatial and temporal control of fundamental biological processes. In addition, access to the expertise and knowledge of the consortium of researchers that have put together this proposal (who have an excellent track record of publications and securing financial support including from the BBSRC) will drive success and ensure maximal impact of the research undertaken. This will support scientific advances and enable technological innovation that will impact all areas of the biological sciences.

The aim of this proposal is to provide a state-of-the-art multimodal imaging platform available to scientific communities across the UoE, other academic institutions throughout the UK, and our partners in industry. The proposal brings together a multidisciplinary consortium of researchers (chemists, biologists, engineers) with expertise in bioimaging with a broad range of diverse research goals. The new platform will therefore underpin scientific research in several key strategic BBSRC priorities for the next decade: combatting microbial resistance; 3Rs in research using animals; sustainably enhancing agricultural production; systems approaches to the biosciences; technology development for the biosciences; synthetic biology.

This proposal aims to upgrade our multimodal coherent Raman-two photon fluorescence (TPF) imaging platform to enhance and extend our imaging capabilities and to foster existing, and develop new collaborations within both academia and industry. We will update our current system using an APE picoEmerald S which uses a fibre laser-based design to improve stability. By using a shorter pulsed system (2 ps, vs 6 ps) a higher peak power is delivered to the sample whilst still retaining a spectrally narrow peak that is ideal for vibrational imaging. This gives up to a 10x improvement in sensitivity in coherent Raman measurements. We will take advantage of the idler output from the laser to add a far-red laser beam to the setup. A number of improvements in optical design will be added, including an improved optical pathway through the microscope to increase transmission. The Olympus XLPLN 25x 1.05 WMP2 lens has been designed with new optical coatings to allow IR transmission allowing far red fluorescence visualisation, whilst the new GaAsP detectors have an improved Quantum Efficiency over multi-alkali detectors allowing for a greater signal to be recovered. Far red fluorophores are popular as in vivo probes due to the enhanced penetration of higher wavelengths into tissue, and the new platform will allow us to incorporate these into our studies and provide increased capability for multiplexing deep in tissues and organs. Two additional Hybrid PMT detectors will be purchased allowing much faster and accurate fluorescence lifetime imaging measurements (FLIM) to be made. By leveraging these recent technological advancements in both laser and microscope design we will be able to significantly expand the functionality and performance we can deliver to researchers including;
(i) Label-free molecular and cellular imaging from cells to animals
(ii) Bioorthogonal imaging of analytes and chemical reactions within tissues
(iii) Development of new imaging probes and technologies

The research underpinned by the investment in this equipment will have far reaching impacts for a variety of research and commercial areas and the wider public.

Academic beneficiaries: as outlined above the academic research output at the UoE will be significantly impacted by acquisition of the new platform. The increased capability and capacity that will be afforded by the new platform will also allow us to foster new academic collaborations both within Edinburgh and beyond. Such projects will be enabled by small pump-priming projects which will be undertaken free of charge.

Industry beneficiaries: we will build on our existing industrial partnerships as well as forging new links with other organisations by leveraging the enhanced capabilities of the new platform and expertise within the consortium. The research outputs will allow development of new products and technologies for market including imaging probes, nanobodies, development of new imaging modalities and metabolic photosensitisers. Working with UK SMEs such as EM Imaging will also allow them to drive forward new innovations and enhance their market position.

Research Students and technical support staff: the new platform will provide training in cutting edge imaging technologies and advanced data analysis tools for both staff and research students. This will impact on the quality of teaching to students across the College of Medicine & Veterinary Medicine and the College of Science and Engineering at the UoE, while equipping our research staff with essential skills for tackling new bioimaging challenges.

Wider impact: the research carried out on the platform will have a wider impact out with the academic and industrial communities by involvement in different public engagement events and STEM activities. In addition, the communications and marketing departments of the University have an excellent record of publicising successful research, in particular in areas of direct relevance to the general public, through press release, social media and media interviews. Members of the consortium also participate in events held at the Scottish Parliament (and through work with research charities), to raise awareness of their research and to impact on government policy making.