Imaging orientational order and lipid density in multilamellar vesicles with multiplex CARS microscopy

Multiplex coherent anti-Stokes Raman scattering (CARS) microscopy is used to measure the width of the orientational order distribution of lipid acyl chains within a three-dimensionally confined microscopic probing volume. A theoretical model is developed to describe and simulate the polarization-dependent measurements. We observe that the orientational order in phosphatidylcholine multilammellar vesicles increases significantly upon addition of small amounts (<or=15 mol%) of cholesterol and is significantly reduced for unsaturated lipids. Based on these measurements and using the quantitative nature of multiplex CARS microscopy the exact local concentration of lipid molecules within the vesicles can be measured in terms of the number of lipid bilayers present in the microscopic probing volume.