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.
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