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

1126312Simon全内反射（TIR）荧光显微镜可对背景极低的膜过程进行高分辨率成像。TIR的独特性质可以使它们自己成为有趣和独特的量化。然而，在对TIR数据进行可靠量化方面存在障碍。首先，由于干涉条纹，用TIR产生的激发场是不均匀的。这些条纹通常比生物信号的幅度更大，是由光源、传递光学器件、物镜和样品中的干涉引起的。这使得荧光强度的定量变得困难。也难以控制激发场偏振，特别是在校正上述干涉条纹时。基于偏振的TIR允许定量测量蛋白质的水平、取向和动力学以及膜取向。这个小组已经表明，极化可以成为探索分子动力学的有力工具。该提案的目标是通过重新设计倏逝场的照明和构建新的显微镜来推进定量TIR成像，以允许：改善TIR激发场的均匀性，控制激发光束，并通过图像采集软件控制激发光束的偏振。

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.