Multiphoton microscopy of lipid-protein dynamics in living cells using correlative Coherent Antistokes Raman Scattering and Two-Photon Fluorescence

The purpose of this research is to develop a new generation optical microscope able to resolve the dynamics of formation, trafficking and breakdown of lipid droplets (LD) and associated proteins in living cells. LD development plays a major role in obesity and diabetes which pose a substantial burden on modern developed countries health budgets. Fluorescence microscopy, using antibodies labelled with dyes or fusion of proteins with fluorescent tags has provided a highly sensitive and specific method of visualizing proteins in cells. Fluorescent markers for optical microscopy of lipids are available, however suffer from rapid photobleaching, that is an irreversible degradation of the fluorescence intensity after excitation with light. Moreover, the cellular modifications arising from the addition of fluorescent lipid probes and lipid staining processes raise major questions if the observed behaviour is real or artefactual. As a result the dynamics of LD development and breakdown has been very difficult to study with fluorescence microscopy, and accurate quantification often impossible. The key idea of the microscope being developed in this project is to obtain the image contrast from lipids via the scattering of light by the vibrations in the lipid chemical bonds (Raman scattering). In this way, the lipids present in cells are visualised without the need of labelling. Raman scattering can be enhanced when using two short laser pulses to excite the vibrations and generate Coherent Antistokes Raman Scattering (CARS). CARS depends nonlinearly on the exciting light intensity, so that sufficient intensities for CARS generation are achieved only in the small focal volume where the exciting photons are concentrated. This results in a very high three-dimensional spatial resolution and lipid droplets of submicron size can be examined with this method. The microscope will be designed to observe lipids on the inside of LDs with CARS, and simultaneously visualise the fluorescence from tagged-proteins associated to the outside of the LD, so that the complete picture of LD development can be determined in living cells. We have identified a number of candidate proteins that have an effect on LDs. This microscope will allow us to investigate the mechanisms behind the interactions of these proteins and LDs in a way that was previously not possible. The identification of the proteins responsible for LD homeostasis in human cells will play a key role in designing therapeutic strategies to control the cellular lipid content. Thus a longer term goal of the project is the development of a high-content screening version of the microscope to discover new targets involved in LDs. Besides the researchers directly involved in this project, several other scientists at Cardiff University are interested in cellular lipids in relation to e.g. atherosclerosis, pulmonary cells producing surfactants preventing lung collapse, breast development and associated milk production. These researchers would greatly benefit from the availability in-house of a CARS/fluorescence microscope dedicated to lipid biology, including a high-content screening version. Results of this work will be published in international journals, so that researchers from both physics and biological disciplines worldwide will benefit from these outcomes. The usage of this microscopy technique will be of relevance in medical applications, for drug discovery and to improve the diagnostic and treatment of lipid-related health problems. Additionally, the proposed research contains the realization of an economic design of the CARS microscope for its possible widespread application. With no CARS microscope commercially available, we expect microscope manufacturers to be interested in its exploitation. Also laser manufacturing companies will be interested in the realisation of laser sources optimised for CARS/multiphoton microscopy. In fact one such company is a collaborator on this project.

Our purpose is to develop correlative Coherent Antistokes Raman Scattering (CARS)/Two Photon Fluorescence (TPF) microscopy to resolve the dynamics of formation, trafficking and breakdown of lipid droplets (LD) and associated proteins in living cells. LD development plays a major role in obesity and diabetes which pose a substantial burden on modern countries health budgets. Fluorescent markers for optical microscopy of LDs are available, yet have a tendency to photobleach rapidly even under moderate illumination. As a result the spatio-temporal dynamics of LD development and breakdown have yet to be determined. In CARS microscopy, the image contrast from lipids is obtained via the scattering of light by the vibrations in the lipid chemical bonds. In this way, lipids intrinsically present in cells are visualised without the need of labelling. CARS is a multiphoton process with very high three-dimensional spatial resolution so that lipid droplets of submicron sizes can be examined. Although CARS microscopy applied to cell biology has been reported in the recent literature, there is no commercially available CARS microscope to date. In this project, we will develop a new generation CARS/TPF microscope based on a single laser, for an economic design, and utilising a differential excitation/detection method to improve CARS image contrast. The microscope will be designed to observe lipids on the inside of LDs with CARS, and simultaneously visualise the fluorescence from tagged-proteins associated to the outside of the LD, for a complete picture of LD development in living cells. We have identified a number of candidate proteins (of the COPI and COPII families) that have an effect on LDs. This microscope will allow us to investigate the mechanisms behind the interactions of these proteins and LDs in a way that was previously not possible. A longer term goal of the project is the development of a high-content screening to discover new targets involved in LDs.

The current first world 'obesity epidemic' is a result of a reduction in physical activity and increase in calorie consumption as changes in modern lifestyle take place. Lipid storage mechanisms contribute to obesity. This project addresses an important shortfall in current methodologies for accurate analysis of the life cycle of a lipid droplet, and the timescale of associated protein interactions. It will have a major impact on basic research in lipid droplet dynamics (within 3-5years) and is likely to feature in clinical diagnosis of lipid storage related genes (5-10yrs). By identifying new mechanisms involved in cellular lipid storage with CARS microscopy, we will discover new targets for the design of therapeutic entities that will enable us to gain control of the cell's lipid storage cycle and improve lipid-related health problems. With no CARS microscope commercially available to date, we expect microscope manufacturers to be interested in the exploitation of the economic CARS microscope design (based on a single laser) developed in this project. Also laser manufacturing companies will be interested in the realisation of sources optimised for CARS/multiphoton microscopy, and one such company is a collaborator in this project. We anticipate markets for the high-content screening CARS technology in commercial drug discovery and therapeutic development, as well as a potential for diagnosis in both public and private sectors (5-10yrs). The contribution of this project to these beneficiaries and to the nation's health, wealth and culture will be mainly through: 1) Knowledge: via the scientific advancements in the understanding of intracellular lipid metabolism and the CARS/multiphoton technology development. 2) People: The RA on this project will receive sate of the art training in photonics. In 2006, Cardiff University established the first MSc Biophotonics course in the UK, jointly taught between the School of Physics and the School of Biosciences, sponsored by microscopy-related industrial collaborators. All applicants teach and/or organize modules on this course. The microscopy technology developed in this project will add to the depth of the research training offered by this course. This will increase the impact and reputation of the course and will allow better recruiting of excellent students to be trained as Biophotonics leaders of tomorrow. 3) Improvement of Health and quality of life. The direct research output will lead to greater understanding of lipid metabolism and reveal new therapeutic targets. We will focus our high content siRNA screen on protein kinases and phosphatases as they make very 'druggable' targets and are currently subject to intensive drug development by companies. This is likely to provide a big reduction in lead time for translation of our research outcome into therapeutic strategies. This gain in cost-effectiveness will ultimately impact on time and development costs, providing a substantial benefit for public health in the prevention and/or cure of lipid-related health problems. Cardiff School of Biosciences has appointed a Head of Innovation, Partnership and Engagement to maximize coordination of commercialization activities. Based on its track record the School is one of three Knowledge Transfer Partnerships (KTP) champions. With direct help from the University KTP team it will run a series of showcases to attract partnerships with companies. Biophotonics is an area of strength of the School, and the outcomes of this research project will be promoted at these events. The School also organises an 'Imaging Symposium' focused on microscopy as a regular annual event which will provide the ideal opportunity to communicate the outcomes of this project to industrial beneficiaries. Links with industrial partners will also occur through MSc projects hosted by companies, and through the initiation of CASE PhD studentships. The School also supports a range of public engagement activities.