A “Molecular Imaging (FLIM/FCS) toolbox” to investigate molecular interactions and activation in super-resolution and widefield mode (360G-Wellcome-202923_Z_16_Z)

Interactions between proteins govern the myriad processes which control cell behaviour. Determining how and where protein complexes form and elicit specific chemical and mechanical signalling events is key to our understanding of the biological processes which underlie development and disease. The ability to analyse signalling events and protein interactions within cells has been enabled by recent technological advances, and we aim to resolve protein complexes and conformations in a wide range of biological contexts. We propose to use a ‘molecular imaging toolbox’, made up of a FLIM/FCS module (coupled to an existing Leica SP8 gSTED) and the recently developed PCO-FLIM camera (interchangeable between several existing platforms e.g. spinning disk, TIRF). This will allow us to image protein interactions and conformations in cells with incredible spatial and temporal resolution and provide new capabilities for several multidisciplinary laboratories to study the molecular basis of how cells sense matrix stiffness and build tissues, and how protein complexes form to dictate cell behaviour during cell fate determination and immune function. These technologies will allow us to effect a step change in our understanding of such complex biological questions, and lead to innovative new discoveries.