超分辨显微成像技术自诞生以来，凭借其优异的纳米级空间分辨率，已成为生命科学研究中精准揭示复杂生命现象的重要成像技术。其中，基于单分子定位的超分辨成像策略，使得定位、观察、研究单个探针分子独特的理、化、光学性能成为可能。偏振作为荧光信号的一个重要特性，近年来伴随着单分子三维取向成像技术的发展，逐步在单分子成像和超分辨领域中展示出诸多新颖且重要的应用特性。本文总结了单分子三维取向超分辨成像技术的最新进展，介绍并分析了两类主要的单分子三维取向荧光显微技术——基于荧光吸收与辐射偏振调制的单分子三维取向成像方法以及利用点扩散函数工程将单个荧光分子的三维取向信息编码到荧光图像上的成像策略。此外，还探讨了应用于活细胞或单颗粒的其他类型的超分辨取向成像技术。最后，针对单分子三维取向超分辨成像技术发展与应用前景面临的挑战，进行了总结与展望。

Super-resolution microscopy with nanoscale spatial resolution has become an important imaging tool in life science research. As a super-resolution technique, single-molecule localization microscopy enables us to localize, identify, and study the unique behaviors of single molecules. At the single-molecule level, the emitted fluorescence signal is highly anisotropic. Resolving the polarization or three-dimensional orientation of single fluorescent molecules is an emerging field in super-resolution microscopy. In this review, we describe the three-dimensional orientation of single molecules through super-resolution imaging techniques. These techniques include fluorescence polarization microscopy and single-molecule orientation imaging through point spread function engineering. Furthermore, we discuss other polarization super-resolution imaging approaches for the applications of live cells and single nanoparticle studies. Finally, we discuss the potential challenges and future research needs of single-molecule orientation localization microscopy. These challenges and requirements can provide in-depth insights into future research in life sciences.