Birefringent Fourier filtering for single molecule coordinate and height super-resolution imaging with dithering and orientation

Super-resolution imaging based on single molecule localization allows accessing nanometric-scale information in biological samples with high precision. However, complete measurements including molecule orientation are still challenging. Orientation is intrinsically coupled to position in microscopy imaging, and molecular wobbling during the image integration time can bias orientation measurements. Providing 3D molecular orientation and orientational fluctuations would offer new ways to assess the degree of alignment of protein structures, which cannot be monitored by pure localization. Here we demonstrate that by adding polarization control to phase control in the Fourier plane of the imaging path, all parameters can be determined unambiguously from single molecules: 3D spatial position, 3D orientation and wobbling or dithering angle. The method, applied to fluorescent labels attached to single actin filaments, provides precisions within tens of nanometers in position and few degrees in orientation. Determining the orientation of single molecules in super resolution imaging is challenging. Here, by adding polarization control to phase control in the Fourier plane of the imaging path, parameters such as 3D spatial position, 3D orientation and wobbling or dithering angle can be determined from single molecules.