提出了一种新思路来确定单分子的三维取向,在全内反射荧光显微成像的基础上,在显微镜的油镜上加一个水层,水和油的折射率不同造成光程差,产生离焦,利用全内反射产生隐失波激发了单分子的纵向分量,并在探测器前增加了一个分光镜,把光路分成偏振方向相互垂直的两束光,使得探测器上的光场分布包含单分子三维取向的信息。推导了隐失场激发的偶极矩光场在探测器上的光强分布表达式,通过分析偏振方向相互垂直的两束光在探测器上的不同的强度分布,来判断单分子偶极矩的三维取向。

A new method is proposed for determination of three-dimensional orientation of the transition dipoles of single molecules. Water layer is added upon the immersion mirror objective on the total internal reflection fluorescence microscopy. The different indices of refraction between water and oil can lead to optical path difference resulting in defocused imaging. A polarization-displacement prism was added before CCD. Beam was split according to the polarization-displacement prism. The field distribution on the CCD includes the information about the orientation of single molecules. Exact field distribution excited by evanescent waves in CCD was considered. The three-dimensional orientation of single molecule was obtained by analyzing the different images of the orthogonal polarization of light.