Super resolution imaging of protein dynamics and functions in physiology and disease

A major challenge in biomedical research is to be able to see inside a cell with enough detail to be able to understand how proteins interact with each other, and what goes wrong in disease. Typically, to see where a protein is inside a cell, a researcher will use a microscope that illuminates the cells with visible light together with dyes to label the proteins. This type of microscope can see detail to about 200nm (1/5000th of a millimetre), but cellular organelles are commonly at least 4 fold smaller than this, and proteins at least 10 fold smaller. Imaging these organelles and proteins with a normal light microscope does not reveal enough detail about the organisation of proteins and organelles within a cell, or help us understand the normal cellular functions of the proteins, and how they are affected by disease. To overcome this problem, several new types of microscopy have been recently invented to help see the detailed organisation of proteins inside cells. The goal of our research is to build a new microscope and develop new dyes that will help us achieve this, and solve important biomedical questions that are impossible to solve with our existing microscopes.

Three main approaches to super-resolution imaging, available as either custom built instrumentation or commercially, are STED (Stimulated Emission Depletion, ~60nm resolution in XY), SIM (Structured Illumination Microscopy, ~100nm resolution in XY) and PALM/STORM (Photo-Activated Localisation Microscopy/Stochastic Optical Reconstruction Microscopy, ~20nm resolution in XY). We are currently building a new microscope (with inward investment from the University) capable of PALM/STORM.
In this new proposal, we plan to build an innovative M2SIM microscope, that will allow multiplanar 3D SIM imaging of live cells as fast imaging rates, to capture high resolution images of dynamic processes inside cells. This will complement the PALM/STORM system by providing high resolution in live cells, at high speeds that are difficult to achieve with PALM. In addition, to maximise the opportunity from our inward investment in PALM, and to serve the general needs of the imaging community, we plan to develop novel caged-dyes and small quantum dots with an increased blinking rate, and develop new approaches to label and tag proteins of interest. Together, these new approaches will allow a range of biomedical researchers at Leeds, working in key strategic areas (Cancer, Cardiovascular, Musculoskeletal, Neuroscience) to answer important biomedical questions that cannot currently be answered using existing technologies.

Beneficiaries for this research will include the microscopy community, and the biomedical science/clinical community. The proposed developments in probes for super-resolution imaging, and the new M2SIM microscope will both be of interest to other researchers using these techniques. The development of both of these technologies is clearly of high interest to other 'imagers' in the UK, as evidenced by the recent Bio-imaging UK meeting at the Wellcome Trust. The participants at that meeting were all very enthusiastic about sharing expertise in these techniques. Through MP's role as chair of the Life Science Committee at the Royal Microscopy Society, she will facilitate workshops to share expertise to ensure that others do benefit. There is additional potential for these approaches being taken up by the commercial private sector, as evidence by our collaboration with Olympus. The biomedical community will benefit from being able to use the technology to answer questions currently impossible to answer with existing standard light microscopy approaches.
These developments have the potential to contribute to the nation's wealth and health, by the value added potential of novel microscopies and their potential for commercialisation, and by the solving of new questions to help us understand important biomedical problems, and devise new approaches to treat disease.
Staff working on the project will be trained in cutting edge techniques, and gain extra professional skills through participating in training others, presenting their work, running workshops, and taking part in other professional development opportunities.
Realistically, by the end of the 5 years, we expect to be running a cutting edge microscopy facility, open to researchers both in Leeds and elsewhere, that we expect will be a flagship for the UK research in this area.