荧光导航冷冻聚焦离子束减薄技术的研究进展  

Recent Developments in Fluorescence-Guided Cryogenic Focused-Ion-Beam Milling

作　　者：李尉兴 卢婧 肖珂 纪伟[1,2,3] Li Weixing;Lu Jing;Xiao Ke;Ji Wei(Key Laboratory of Interdisciplinary Research,Institute of Biophysics,Chinese Academy of Sciences,Beijing 100101,China;Innovative Development Center of Light Microscopy and Detection Technology,Bioland Laboratory,Guangzhou 510320,Guangdong,China;College of Life Science,University of Chinese Academy of Sciences,Beijing 100049,China)

机构地区：[1]中国科学院生物物理研究所交叉科学所重点实验室,北京100101 [2]生物岛实验室光学成像和检测技术研发创新中心,广东广州510320 [3]中国科学院大学生命科学学院,北京100049

出　　处：《中国激光》2023年第21期28-41,共14页Chinese Journal of Lasers

基　　金：国家自然科学基金(62105356);国家杰出青年科学基金(T2225020)。

摘　　要：细胞超微结构的原位解析是当前的一个研究热点。冷冻电子断层扫描成像技术(cryo-ET)是目前细胞原位结构解析的核心技术。cryo-ET只能对厚度小于300 nm的样品进行成像,因此利用cryo-ET研究细胞超微结构时首先需要对细胞进行减薄。聚焦离子束(FIB)切割是目前冷冻生物样品减薄的主流技术。传统FIB切割只能在细胞的任意位置上进行“盲切”,无法对细胞内部特定研究目标进行定点切割。光电融合成像技术(CLEM)恰可解决这一问题。CLEM利用荧光成像技术识别并定位研究目标,通过光电图像的关联匹配,可在FIB图像中确定荧光目标的位置,进而指导FIB的定点减薄。针对荧光导航cryo-FIB减薄的相关技术方法、仪器设备和工作流程进行了梳理,分析对比了主流方案的优缺点,旨在帮助研究者选择出合适的荧光导航FIB减薄方案,并对该技术的未来发展方向进行了展望。Significance Eukaryotic cells have numerous cellular structures,including a variety of organelles and macromolecular complexes.These structures have specific physiological functions and work interactively to perform certain cellular activities.Therefore,studying these structures in their native state is essential to understand the real physiological processes in the cells.In situ investigation of cellular structures does not only provide morphology,distribution,and abundance information,but also reveals their interaction mechanisms,thereby providing new insights into the understanding of life.Cryo-electron tomography(cryo-ET)is currently the principal technique to resolve the in situ structures of biological specimens.By collecting tilted series of transmission electron images and performing image reconstruction,cryo-ET determines the 3D structures of bio-specimens with a nanometer-level resolution.A prerequisite for applying cryo-ET is to fix the sample under cryogenic conditions.High-pressure freezing and plunge freezing are well-established cryo-fixation methods that preserve biological specimens in their near-native state in vitreous ice.Benefiting from these techniques,cryo-ET has been widely applied to cells and tissues.One limitation to cryo-ET is its restricted imaging depth,which is typically a few hundred nanometers owing to the confined penetration capabilities of electrons.Therefore,reducing the thickness of the samples to that of lamellae of approximately 200 nm is necessary before applying cryo-ET.Focused ion beam(FIB)milling has been recently employed to prepare lamellae of bio-specimens for cryo-ET.Compared to traditional ultramicrotomy,FIB milling avoids artifacts such as distortions,crevasses,and compression when fabricating the lamella.However,conventional FIB milling does not allow site-specific milling,because in a dual-beam FIB/SEM system,FIB or SEM image only illustrates the surface morphology of the sample and cannot provide more information to recognize and localize the underlying interes

关 键 词：生物医学 冷冻电子断层扫描成像技术 扫描电镜 荧光成像技术 快速冷冻 聚焦离子束减薄 光电融合成像技术 

分 类 号：Q-31[生物学]