Fatty acid (FA) intake and its subsequent fate are implicated the two most prevalent diseases in the developed world, coronary heart disease and type 2 insulin-resistant diabetes. More than 300 million people world-wide live with type 2 diabetes, and its incidence is increasing rapidly, especially in developing countries. In Europe alone, direct medical expenses for type 2 diabetes totals more than 30 Billion € per year and more than 50% of these costs are related to hospitalization. This Career Integration Grant application outlines the PI’s research programme to use a non-linear vibrational imaging technique in order to explore the underlying FA dynamics during ectopic fat deposition and FA metabolism in muscle cells.Ectopic fat appears in muscle of insulin-resistant, type 2 diabetics and also, paradoxically, in endurance trained athletes – who have acute insulin sensitivity. The methods developed in this proposal will provide a platform for answering many unresolved questions regarding FA dynamics in muscle cells from intake (and lipogenesis) to FA metabolism in both normal and insulin-resistant muscle cells. The scientific results gleaned from this research will provide new data about FA packaging into LDs and FA metabolism upon triggered lipolysis as a function of insulin activity levels. Furthermore, experiments investigating LD proximity to mitochondria in normal and insulin-resistant muscle cells will provide much needed data to help unravel the “athletes paradox” outlined above. The outcome of the PI’s research plan will reveal a link between FA processing and insulin resistance, which is critical for design of new therapeutics to treat type 2 diabetes.