Enhanced 4Pi single-molecule localization microscopy with coherent pupil based localization and light sheet illumination

Over the last decades, super-resolution techniques have revolutionized the field of fluorescence microscopy. Among them, interferometric or 4Pi microscopy methods exhibit supreme resolving power in the axial dimension. Combining with single-molecule detection/localization and adaptive optics, iPALM/4PiSMS/W-4PiSMSN allowed 10-15 nm isotropic 3D resolution throughout the whole cell. However, further improving the achieved 3D resolution poses significantly challenges which, in part, is blocked by the complexity of single-molecule emission pattern generated by these systems rendering a large portion of information carrying photons unusable. Here we introduce a localization algorithm that achieves the theoretical information limit for 4Pi based single-molecule switching nanoscopy (4Pi-SMSN), and demonstrates improvements in resolution, accuracy as well as applicability comparing with the state of art 4Pi-SMSN methods. Further, with a novel 4Pi-compatible light-sheet illumination reducing the fluorescence background by >5-fold, we demonstrated the new system enables further improvement in the achievable resolution of 4Pi/interferometric single-molecule imaging systems.