Abstract Advances in single-molecule localization microscopy are providing unprecedented insights into the nanometer-scale organization of protein assemblies in cells and thus a powerful means for interrogating biological function. However, localization imaging alone does not contain information on protein conformation and orientation, which constitute additional key signatures of protein function. Here, we present a new microscopy method which combines for the first time Stochastic Optical Reconstruction Microscopy (STORM) super-resolution imaging with single molecule orientation and wobbling measurements using a four polarization-resolved image splitting scheme. This new method, called 4polar-STORM, allows us to determine both single molecule localization and orientation in 2D and to infer their 3D orientation, and is compatible with high labelling densities and thus ideally placed for the determination of the organization of dense protein assemblies in cells. We demonstrate the potential of this new method by studying the nanometer-scale organization of dense actin filament assemblies driving cell adhesion and motility, and reveal bimodal distributions of actin filament orientations in the lamellipodium, which were previously only observed in electron microscopy studies. 4polar-STORM is fully compatible with 3D localization schemes and amenable to live-cell observations, and thus promises to provide new functional readouts by enabling nanometer-scale studies of orientational dynamics in a cellular context.

4polar-STORM polarized super-resolution imaging of actin filament organization in cells