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.