新型高分辨率光电关联成像技术的研究及其在细胞生物学中的应用

Correlative light and electron microscopy (CLEM) offers a means of guiding the search for the unique or rare events by fluorescence microscopy (FM) and allows electron microscopy (EM) to zoom in on them for subsequent EM examination in three-dimensions (3D) and with nanometer-scale resolution. FM visualizes the localization of specific antigens by using fluorescent tags or proteins in a large field-of-view to study their cellular function, whereas EM provides the high level of resolution for complex structures. And, cryo-CLEM combines the advantages of preserving structures in a near-native state throughout the entire imaging process and by avoiding potentially harmful pre-treatments, such as chemical fixation, dehydration and staining with heavy metals. In recent years, many new instruments and software which intended to optimize the workflow and to obtain better experimental results of CLEM have been presented or even commercialized. However, the specimen damage during transfer from FM to EM and the resolution of FM still need to be improved..In the past two years, we have successfully developed a High-vacuum Optical Platform to develop CLEM imaging technology (HOPE), which was designed to realize high-vacuum optical ( fluorescent) imaging for cryo-sample on EM cryo-holder (e.g. Gatan 626). A non-integrated high-vacuum optical stage, which adapted to the EM cryo-holder, was fixed on an epi-fluorescence microscope to obtain fluorescent images. And then the EM cryo-holder was transferred to EM for collection of EM data. This protocol was aimed to minimize the specimen damage during transfer from FM to EM and it was versatile to expend to different types of light microscopy or electron microscopy..Based on the past work, we intend to optimize the CLEM workflow and combine SRRF (super-resolution radial fluctuations) High-Resolution(HR) Microscopy in HOPE (HR-HOPE) to increase the fluorescence imaging resolution and correlative accuracy. For this project, CLEM based on HR-HOPE will guide the search for the mitochondrial fission events by fluorescence microscopy and record 3D ultrastructural details provided by cryo-electron tomography (ET). The project includes: (1)The development of HR-HOPE; (2)The sample preparation and workflow of CLEM based on HR-HOPE; (3)Cryo-ET 3D reconstruction of mitochondrial fission in Neuro-2a cells.

随着对细胞生物学的深入研究，如何精确示踪胞内重要生命现象并获取高分辨结构信息已经成为当前学科发展的重点攻关方向之一。光电关联成像技术可以实现荧光特异标记定位与电镜高分辨结构解析的优势整合，日渐受到广泛关注。然而现有光电关联成像装置难于在使用便捷性和关联精确度上获得统一，大大制约了该技术在生物学中的应用。.前期工作中，我们已经完成了一套基于高真空冷台的便捷式光电关联成像装置，在此工作基础上，本项目拟结合高分辨荧光显微成像技术（SRRF, super-resolution radial fluctuations），开发新型高分辨率光电关联成像技术并探究其在细胞生物学中的应用。主要研究内容：（1）基于高真空冷台的高分辨率光电关联成像系统的总体设计；（2）冷冻光电关联成像样品制备及技术流程优化；（3）以小鼠神经瘤细胞作为测试样品，荧光标记示踪胞内线粒体形态变化，完成高分辨率电镜数据采集与三维重构。

冷冻光电关联成像技术可以实现荧光特异标记定位与电镜高分辨结构解析的优势整合，然而现有光电关联成像装置难于在使用便捷性和关联精确度上获得统一，大大制约了该技术在生物学中的应用。本项目在项目组前期基于高真空冷台的冷冻光电关联技术工作基础上，结合结构光照明高分辨荧光显微成像技术，建立了一套基于结构光照明的高分辨率冷冻光电关联技术流程，完成了系统硬件设计以及系统集成控制，实现了真空环境下基于长工作距离物镜的冷冻三维结构光照明高分辨率成像，其中冷冻光学成像横向分辨率可达190nm（传统的冷冻宽场荧光成像分辨率约400nm），纵向分辨率可达600nm；同时，项目组编写并优化了一款三维冷冻光电关联软件3D-View，该软件目前可以实现冷冻荧光-透射电镜关联匹配（关联定位平均偏差约51nm）及冷冻荧光-扫描电镜关联匹配（平均偏差约150nm）。基于以上研究成果，项目组以荧光标记线粒体的小鼠神经瘤细胞和海拉细胞，以及疱疹病毒感染的叙利亚幼地鼠肾细胞样品作为测试对象，对荧光信号指示区域完成三维冷冻结构光照明高分辨率成像，通过光电关联软件进行高精度匹配，指导后续聚焦离子束对目标区域进行减薄，获得厚度在200nm以内的包含目标结构的冷冻含水切片样品后，送入冷冻透射电镜中对目标结构进行成像，高效地获得了正在分裂的线粒体以及胞内的病毒颗粒的冷冻电镜原位结构。实测结果证明，本项目研制成功的高分辨率冷冻光电关联成像技术有助于解决原位结构生物学研究中缺少精准定位目标区域的技术难题，为研究胞内微米乃至数百纳米超微结构，例如线粒体、疱疹病毒等的原位超微形态研究提供一种更为高效、精确又直观的研究手段。