3D optical nanoscopy with excited state saturation at liquid helium temperatures

We present a 3D fluorescence nanoscopy method operating at cryogenic temperatures, based on optical saturation of the excited state of individual molecules. Using a focused laser beam structured with a zero-intensity central region surrounded by intensity gradients in the three space directions, we achieve a sub-30 nm 3D optical resolution. Moreover, the analysis of the fluorescence scanning images of single molecules reveals the 3D orientation of their transition dipole with an accuracy of a few degrees. This method provides a valuable tool for locating neighboring molecules with overlapping optical transitions in order to study their interactions.