Enhanced 4Pi single-molecule localization microscopy with coherent pupil based localization

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. Combined with single-molecule detection/localization and adaptive optics, current 4Pi microscopy methods enabled 10–15 nm isotropic 3D resolution throughout whole cells. However, further improving the achieved 3D resolution poses challenges arising from the complexity of single-molecule emission patterns generated by these coherent single-molecule imaging systems. These complex emission patterns render a large portion of information carrying photons unusable. Here, we introduce a localization algorithm that achieves the theoretical precision limit for a 4Pi based single-molecule switching nanoscopy (4Pi-SMSN) system, and demonstrate improvements in localization precision, accuracy as well as stability comparing with state-of-the-art 4Pi-SMSN methods.

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.