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

光学显微镜在可视化肉眼无法辨别的生物方面起到了重要作用。然而，它观察小于200 nm的物体的能力是有限的。超分辨率光学显微镜就是为了克服这一局限而发展起来的。建议的研究集中在一种特殊的超分辨率显微镜方法，称为单分子定位显微镜(SMLM)。在这项技术中，一次激活一小部分荧光团，并收集它们的点状图像并在数学上进行定位。在积累足够的定位坐标后，就可以重建出超分辨率的图像。本研究将致力于通过结合新的方法来提高SMLM的分辨率。该提议的第一个目标是通过在低温下成像并结合使用镜子增强荧光来提高2D分辨率。第二个目标是通过将低温成像与4PI显微镜相结合来提高3D分辨率。4PI显微镜是一种使用两个可移动的物镜来收集更多光线的成像方式。最后，将单粒子重建结合到图像分析中。这是一种对同一粒子的许多图像进行计算对齐和平均的技术，从而提高了分辨率。这些新的成像方法将被用来原位了解核孔复合体的结构和功能。此外，本研究有望推进SMLM在理解生物分子原位结构和功能方面的适用性，从而为结构生物学提供一种新的方法。

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.