The orientation of fluorophores can reveal crucial information about the structure and dynamics of their associated subcellular organelles.Despite significant progress in super-resolution,fluorescence polarization microscopy remains limited to unique samples with relatively strong polarization modulation and not applicable to the weak polarization signals in samples due to the excessive background noise.Here we apply optical lock-in detection to amplify the weak polarization modulation with super-resolution.This novel technique,termed optical lock-in detection super-resolution dipole orientation mapping (OLID-SDOM),could achieve a maximum of 100 frames per second and rapid extraction of 2D orientation,and distinguish distance up to 50 nm,making it suitable for monitoring structural dynamics concerning orientation changes in vivo.OLID-SDOM was employed to explore the universal anisotropy of a large variety of GFP-tagged subcellular organelles,including mitochondria,lysosome,Golgi,endosome,etc.We found that OUF(Orientation Uniformity Factor) of OLID-SDOM can be specific for different subcellular organelles,indicating that the anisotropy was related to the function of the organelles,and OUF can potentially be an indicator to distinguish normal and abnormal cells (even cancer cells).Furthermore,dual-color super-resolution OLID-SDOM imaging of lysosomes and actins demonstrates its potential in studying dynamic molecular interactions.The subtle anisotropy changes of expanding and shrinking dendritic spines in live neurons were observed with real-time OLID-SDOM.Revealing previously unobservable fluorescence anisotropy in various samples and indicating their underlying dynamic molecular structural changes,OLID-SDOM expands the toolkit for live cell research.