MRI: Development of Polarization Control for Total Internal Reflection Fluorescence Microscopy

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1126312SimonTotal internal reflection (TIR) fluorescence microscopy allows high resolution imaging of membrane processes with very low background. Unique properties of TIR can lend themselves to interesting and unique quantifications. However there are obstacles in the way of robust quantification of TIR data. First, the excitation field created with TIR is non-homogenous due to interference fringes. These fringes, which are often of larger magnitude than the biological signals, result from interferences in the light source, the delivery optics, the objectives and the sample. This makes quantification of fluorescence intensities difficult. It is also difficult to control the excitation field polarization, particularly while correcting for interference fringes discussed above. Polarization based TIR allows for quantitative measurements of the levels, orientation and dynamics of proteins as well as membrane orientations. This group has shown that polarization can be a powerful tool for exploring the dynamics of molecules. The goal of this proposal is to advance quantitative TIR imaging by redesigning the illumination of the evanescent field and building a new microscope to allow: Improved uniformity of the TIR excitation field, control of the excitation beam, and through the image acquisition software control of the polarization of the excitation beam.