Understanding the way proteins are organized in cells is an important element to answer key questions in biology. Super resolution fluorescence imaging has brought a considerable step in this direction; Stochastic optical reconstruction microscopy (STORM) and Photoactivation light microscopy (PALM) are able to provide a spatial resolution down to tens of nanometers, exploiting the high precision of single molecule’s point spread function (PSF) localization, even in dense samples. While these methods measure single molecule’s positions, they however do not give access to their orientation, which is required to investigate organization, alignment and conformational changes. Reporting single molecule’s orientation, at the same time as their position, is challenging : in this workshop we will describe and demonstrate different approaches to solve this problem, based on polarized fluorescence imaging and splitting of polarized detection channels [1] or PSF engineering [2]. Combining orientation and localization super resolution imaging opens new directions towards structural imaging of complex proteins assemblies in cells. We will discuss how to access to 3D orientation information based on these schemes. [1] C. Rimoli, C. Valades Cruz, V. Curcio, M. Mavrakis, S. Brasselet. 4polar-STORM polarized super-resolution imaging of actin filament organization in cells. Nat. Communications 13, 301 (2022) [2] V. Curcio, et al. Birefringent Fourier filtering for single molecule Coordinate and Height super-resolution Imaging with Dithering and Orientation (CHIDO), Nat. Communications 11 (1) (2020)