The objective of this project is to develop coherent anti-Stokes Raman scattering (CARS) microscopy to study membrane rafts. The PI will develop three-color CARS microscopy in which two images, one tuned to the peak and the other tuned to the dip of a CARS band, are acquired simultaneously. The difference of the two images will permit high-sensitivity vibration imaging of single membranes. The resonant CARS signal from the aliphatic C-D vibration will be used to determine the partition ratio of a specific lipid between the liquid ordered (rafts) and disordered domains. The PI will investigate whether rafts are formed by an interaction between cholesterol and lipid hydrocarbon chains or by hydrogen bonding between cholesterol and sphingomyelin. He will use the polarization sensitivity of CARS to determine the orientation of the CH2 groups of lipid hydrocarbon chains in a bilayer containing cholesterol. This method will be able to measure the ordering degree of lipids in rafts. The PI will also develop coherent Raman microspectroscopy for spectral characterization of lipid-lipid characterization in single membrane domains. The PI will directly test the hypothesis of lipid rafts in the plasma membrane of live cells. Aggregation of fluorophore labeled rafts has been observed in different cellular processes. He will examine if these macroscopic domains are rich in sphingomyelin and cholesterol using CARS microscopy. The non-resonant background that arises from water and other cellular organelles will be removed by using the three-color excitation scheme.
This project provides an environment for interdisciplinary scientific training. Students involved in this research will need to combine the knowledge in chemistry, physics, and biology. The new imaging methods developed in this project will be incorporated in the PI's graduate level course of Biomedical Optics and undergraduate level course of Senior Engineering Design.
