Fig. 1. Schematic of SMFP technique^[20]

Fig. 2. Single-molecule localization affected by molecular orientation. (A) Single-molecule fluorescence with a fixed orientation forms an asymmetric, elongated three-dimensional PSF in the image space, where slight defocus can cause a lateral shift in the photon distribution relative to the true molecular position^[30]; (B) schematic of 4f system^[33]; (C) the “y-phi” mask, which converts square polarization light into y-polarized light; (D) schematic diagram of the “y-phi” mask converting radial polarization light into x-direction and converting azimuthal polarization light into -y direction BFP and PSF simulation, scale bar: 200 nm^[31]

Fig. 3. Schematic diagrams of single-molecule three-dimensional orientation^[38] and CHIDO technique^[39]. (A) Azimuthal angle φ, polar angle θ, and solid angle Ω; (B) back-focal plane images of RHC and LHC; (C) the PSFs and CHIDO PSFs at the nominal focal plane z = 0 (top) and at z = 300 nm (bottom), corresponding to five different dipole orientations; (D) single-molecule three-dimensional localization and orientation imaging of F-actin filaments

Fig. 4. Schematic of Vortex PSF^[42]. (A) Microscope with a vortex phase plate generates asymmetric donut-like shape, Vortex PSF, for fixed emitters on the camera; (B) phase profile of the vortex phase plate; (C) PSF obtained without using a vortex phase plate for dipoles of different orientations; (D) Vortex PSF formed by dipoles of different orientations using a vortex phase plate

Fig. 5. Super-resolution SMOLM imaging of lipid nanodomains^[38]. (A) Tri-spot and Duo-spot phase masks; (B) Tri-spot and Duo-spot PSFs, scale bars, 2 µm (left) and 500 nm (right); (C) Nile red orientation with different cholesterol levels; (D) Nile red orientation with various acyl chain structures; (E) SMOLM imaging of SLB nanodomains, scale bars, 2 µm

Fig. 6. Principle for polarized vortex imaging^[45]. (A) schematic of the optical setup; (B) representative dipole orientations (top) and corresponding polarized vortex PSFs (bottom), scale bar, 300 nm; (C) molecular binding orientations and rotational diffusion on an amyloid fibrillar network, scale bar, 1 µm

Fig. 7. raPol microscope^[50]. (A) VWP placed at the back focal plane of the imaging system; (B) representative radially (red) and azimuthally (blue) polarized PSFs of rotationally fixed molecules, scale bar, 500 nm; (C) detection and orientation estimation performance of raPol and xyPol PSFs; (D) rotational and translational diffusion of single molecules with increasing cholesterol concentration, scale bar, 50 nm

Fig. 8. raMVR microscope^[47]. (A) Schematic of the raMVR setup; (B) the pyramidal and air pyramid mirrors in raMVR; (C) raw images projected on the detectors, scale bar, 20 µm; (D) representative DSF in each channel for one molecule, scale bars, 500 nm; (E) 6D SMOLM images of lipid-coated spheres with with radii of 150 nm, 350 nm and 1,000 nm; (F) 6D SMOLM images of HEK-293T cell, scale bar, 2 µm