Stem cell science and technology hold a great deal of promise within the context of tissue repair and regeneration. Whilst we know a lot about the function and response of stem cells in the laboratory, we know very little about their behaviour in tissues within individuals, delaying their applicability to the clinics. A major barrier has been the inability to accurately track cell lineages and to distinguish them from other cell types within the tissue. Fundamental questions, i.e. when one cell divides to become two cells are they the same or different? is this effect the same or different each time the cell divides?, have not been answered yet. The proposed project will address these issues by developing microscopic techniques that allow us to image these cells in a non-invasive, non-harmful way (unlike current approaches) and we will utilise technologies that will eventually enable us to track these cells deep within patient tissues. Coherent Anti-Stokes Raman Scattering (CARS) has recently emerged as a new multiphoton microscopy technique which directly addresses endogenous biomolecules with high chemical specificity without the need for exogenous labelling. This imaging technology would be ideal to follow non-invasively, living stem cells during differentiation in real time at physiological conditions without any of the artefacts/problems associated with labelling. We will use different modalities to identify endogenous markers of stem cell differentiation for tracking purposes. First, CARS and Differential CARS will be used to examine the lipid distributions within stem cells undergoing differentiation. Furthermore, to improve the chemical specificity we will develop a novel Coherent Multiplex CARS method to address a large number of vibrational frequencies simultaneously. In this way tracking of stem/progenitor cells and their progeny in vivo without the need for fluorescent, harmful labels or labels that are lost as the cell divides will be possible.