Study of Membrane Domains with Coherent Raman Microscopy and Microspectroscopy

本项目的目的是开发相干反斯托克斯拉曼散射（CARS）显微镜来研究膜筏。PI将开发三色CARS显微镜，其中两幅图像，一幅调到CARS波段的峰值，另一幅调到最低点，同时获得。这两个图像的差异将允许对单个膜进行高灵敏度振动成像。来自脂肪族C-D振动的共振CARS信号将用于确定液体有序（筏）和无序结构域之间特定脂质的分配比率。PI将调查筏是由胆固醇和脂质碳氢链之间的相互作用形成的，还是由胆固醇和鞘磷脂之间的氢键形成的。他将利用CARS的极化敏感性来确定含有胆固醇的双分子层中脂质碳氢链的CH2基团的取向。该方法将能够测量筏中脂质的有序度。PI还将开发相干拉曼微光谱，用于单膜域脂质表征的光谱表征。PI将直接检验活细胞质膜中脂筏的假设。在不同的细胞过程中观察到荧光团标记筏的聚集。他将使用CARS显微镜检查这些宏观结构域是否富含鞘磷脂和胆固醇。由水和其他细胞器产生的非共振背景将通过使用三色激发方案去除。这个项目为跨学科的科学训练提供了一个环境。参与这项研究的学生需要将化学、物理和生物学的知识结合起来。本项目开发的新成像方法将被纳入PI的生物医学光学研究生课程和高级工程设计本科课程。

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.