Single particle cryogenic super-resolution microscopy solves multi-protein structures of nuclear pore complexes

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Light microscopy has been instrumental in visualizing biology indiscernible by eye. However, it is lim- ited in its ability to observe objects smaller than 200 nm. Super-resolution light microscopy has been developed to overcome this limitation. The proposed research focuses on a particular super-resolution microscopy method called single-molecule localization microscopy (SMLM). In this technique, a small subset of fluorophores are activated at one time, and their point-like images are collected and localized mathematically. After accumulating enough localization coordinates, a super-resolution image can be reconstructed. This research will focus on enhancing the resolution of SMLM through combinations of novel methods. The first aim of the proposal is to increase the 2D resolution by imaging at cryogenic temperatures in combination with enhancing fluorescence with a mirror. The second aim is to increase 3D resolution by combining cryogenic imaging with 4Pi microscopy - an imaging modality using two op- posing objective lenses to collect more light. Finally, single particle reconstruction will be incorporated in image analysis. This is a technique that computationally aligns and averages many images of the same particle, thereby boosting resolution. These novel imaging methods will be used to understand nuclear pore complex structure and function in situ. Beyond this, this research is expected to advance the applicability of SMLM to understanding the in situ structure and function of biological molecules, thereby providing a new method to structural biology.