Multiparametric advanced fluorescence imaging strategies for in situ analysis of live cell signalling

To understand and combat the causes of human disease, we must understand the basic structure and function of the individual cells that make up the tissues and organs of the human body. For example, to allow the design of effective therapies to target cancer we first need to answer fundamental questions about how the growth, division and movement of cells are controlled. Robert Hooke was the first to use microscopes to describe cell structure in 1665, and since then microscopy has become one of the most powerful tools for cell biologists across the world. The power of light microscopes has of course continued to increase since their invention but, remarkably, the most dramatic improvement has come in the last ten years or so. In that period physicists have worked out how to measure the location of a single protein in a cell with a precision about ten times better that was previously thought possible. This is important because we can now see the internal structure and organisation of cells in much more detail. In parallel, physicists working together with biologists developed microscopical methods that, instead of just producing a map of the locations of one particular protein inside a cell, can produce a map of precisely where protein A is bound to protein B. This is a fundamental advance, because cell function is controlled by pathways and networks of such interactions between specific proteins. Potentially then, these new microscopes provide a window into the internal workings of a cell that allow us to see these protein networks. However, at the moment, the most detailed images can only be obtained from chemically preserved rather than living cells, and each image takes many minutes to record. This is a serious problem, because the interactions between proteins that control cell function take place transiently on the time scale of seconds. To understand cell function, we need movies rather than still images. In the present proposal, biologists and physicists will work together to develop the technology to allow us to record the detailed maps of protein locations and interactions in live cells in milli-seconds rather than minutes or hours. We think that these new developments will unlock the potential of these microscopes to show us how cells work at the molecular level.

The power of optical imaging technology to drive major discoveries in cell biology and medicine has increased dramatically over the last decade. This proposal focuses on two areas of fluorescence microscopy where the potential for such applications is clear but has not yet been fully realised. The first relates to functional imaging modalities that can map specific protein-protein interactions in cells, in particular using Forster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM). The second relates to super-resolution microscopy (SRM) methods that break the conventional resolution limit imposed by the wavelength of light. In recent years, many different SRM technologies have been developed that typically promise a spatial resolution of 50 nm, an order-of-magnitude improvement over conventional methods. However the commercially available FLIM and SRM instruments are limited by technical constraints of sub-optimal detector sensitivity, speed, data analysis and interpretation. We will develop new FLIM and SRM configurations and detectors to overcome these limitations, and allow protein location, orientation, environment, interactions and dynamics to be analysed in living cells and organisms. These instruments will all be built within a new Microscopy Development Centre (MDC) on the Guy's Hospital Campus of King's that is adjacent to the new Nikon Imaging Centre. These two Centres will share technical support, training, data storage and image analysis facilities/expertise. The MDC physicists will work closely with biomedical scientists in the co-I's teams to refine the new instruments and apply them to a series of exemplar biological questions in the fields of immunology, stem cell biology, cancer, cardiovascular and muscle biology. Once developed, the new FLIM and SRM technologies will be disseminated within the biomedical research community, initially within King's and subsequently to other UK Universities, institutes and companies.

This proposal represents a core multidisciplinary partnership between academics with very strong links to commercial collaborators. Our goal is to design and build novel instruments to analyse direct protein-protein interactions and image three-dimensional complexes, structures and dynamic processes in living cells at the nanometer scale to reveal previously hidden details of biological structure and function. The proposed multidisciplinary partnership between several laboratories has been formed through a shared common goal to use novel imaging approaches to unravel complex biological signalling events in detail in living cells or organisms. The partnership brings together biologists, biophysicists and physicists within an environment that is ideal for training, technology development and image analysis. The team already has a track record of joint publications and research funding, as well as group meetings, journal clubs and joint PhD student supervision. The significant advances that are made in microscopy development here will eventually also be applied across multiple laboratories with different biological questions and thus provide an innovative and collaborative training environment that will broaden the knowledge base of our physics and biology postgraduate/postdoctoral staff as well as the participating faculty. We anticipate that the programme of novel optical instrument development detailed here will also have a significant impact on the research community at King's and more broadly within the UK. The alignment of King's College London with partner NHS Hospitals in King's Health Partners (KHP) also offers unparalleled opportunities for translational and clinical research. King's is also a partner in the Francis Crick Institute (FCI), and as the biophysical/medical interface is a key strategic development area of the FCI, the state-of-the-art developments made possible through our partnership will also likely extend to collaborations in the FCI in future. For the current proposal, we have established collaborations with Universities of Sussex and Edinburgh who will benefit directly from the technology arising from this project for their own research purposes. We additionally propose to host a conference/workshop in year5, at the end of the current proposed development plan, to share our findings and progress with the national and international research community. Microscopy instrument development represents one of the growing technology areas in biomedicine and our existing strong working relationship with imaging instrument manufacturers places us in an excellent position to fully realize the commercial potential of our novel developments arising from this study. Similarly, the importance of microscopy-based assays and screens to analyse cell function and test potential drugs is increasingly recognised by the pharmaceutical industry and will place us at the forefront of emerging technologies leading to the design and validation of new therapies.