有机荧光团及荧光标记细胞的阴极荧光成像研究  

Cathodoluminescence Imaging of Organic Fluorophores and Fluorescently Labeled Cells

作　　者：李长硕 李硕果[2] 季刚[2] 王丽[1] 李劲涛[3] 王莉[2] 贾星 张小云 韩晓东[1] 吉元[1] 孙飞[2,4] LI Chang-Shuo;LI Shuo-Guo;JI Gang;WANG Li;LI Jin-Tao;WANG Li;JIA Xing;ZHANG Xiao-Yun;HAN Xiao-Dong;JI Yuan;SUN Fei(Faculty of Materials and Manufacturing,Beijing University of Technology,Beijing 100124,China;Center for Biological Imaging,Core Facilities for Protein Science,Institute of Biophysics,Chinese Academy of Sciences,Beijing 100101,China;Faculty of Environment and Life,Beijing University of Technology,Beijing 100124,China;Bioland Laboratory,Guangzhou 510005,China)

机构地区：[1]北京工业大学材料与制造学部,北京100124 [2]中国科学院生物物理研究所蛋白质科学研究平台生物成像中心,北京100101 [3]北京工业大学生命科学与生物工程学院,北京100124 [4]生物岛实验室,广州510005

出　　处：《生物化学与生物物理进展》2023年第8期2006-2017,共12页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金(11604006,91860202,31770999);北京结构生物学高精尖创新中心课题(997012045-100300001);北京市杰出青年科学基金(BJJWZYJH01201910005018);北京市自然科学基金(Z180014);北京教委科学基金(KM201910005004);中国科学院仪器设备功能开发技术创新项目(2021gz015)资助。

摘　　要：目的阴极荧光(CL)成像是一种以电子束为激发源的高分辨荧光成像技术,但生物材料对电子束的敏感性限制了CL技术在生命科学中的广泛应用。为了研究和发展CL技术在生物样品中的应用,本文旨在通过探究电子辐照引起碳基材料的结构损伤、有机基团的降解及荧光猝灭等问题,深入理解电子源对有机荧光团的激发特性。方法本研究应用扫描电镜(SEM)和阴极荧光谱仪系统(SEM-CL),研究电子源对有机荧光团及荧光探针标记细胞的激发特性,观测了有机物的CL信号的发射特性、强度衰减、成像方式及特点。结果实验结果显示,在低能量(2.5~5 keV)和低束流(~10 pA)电子辐照下,有机荧光微珠发射出较强的荧光,CL像分辨率达到~30 nm。荧光微珠经过12 min辐照,信号强度衰减了25%,CL像仍保持了可接受的发光强度和足够的信噪比。此外,还获得了从细胞表面到内部一定深度内,荧光标记的亚细胞结构信息。结论在SEM-CL系统中,可以同时获得由电子束激发产生的电子像和CL像,实现阴极荧光与电子显微镜关联(CCLEM)成像。本实验的研究结果为CCLEM技术应用于生物结构研究提供了数据及技术支持。Objective The correlative cathodoluminescence(CL)imaging is a high-resolution fluorescence imaging technique using electron beam as excitation source.However,the sensitivity of biomaterials to electron irradiation limits the wide application of CL technique in life science.In order to study and develop the application of CL technology in biological samples,this paper aims to deeply understand the excitation characteristics of organic fluorophore by electron source by exploring the structural damage of carbon substrate,degradation of organic groups and fluorescence quenching caused by electron irradiation.Methods We used a CL and electron microscopy(CCLEM)technique in a field emission scanning electron microscope(SEM)to observe organic fluorophores and fluorescently labeled cells.Results We studied excitation and emitting characteristics of organic fluorophores by electron beam.Under the irradiation of low energy(2.5-5 keV)and low current(~10 pA),the fluorescent microbeads had the stronger and stable CL emission,the resolution of CL image reached~30 nm.After 12 min irradiation,fluorescence intensity of microbeads decreased by 25%,and CL image still maintained acceptable fluorescence intensity and adequate signal to noise ratio.Further,fluorescently labeled subcellular structures were identified from cell surface to a certain depth inside the cell.The nuclei and organelles were well observed with a bulk-sample imaging setup,while membrane proteins,that are widely localized on the cell surface,were clearly identified with a home-made imaging setup of thin-sample.Conclusion Results provide data and technical support for the application of CCLEM in the study of biological structures.CCLEM bioimaging can also be used as an important supplement to correlative light and electron microscopy(CLEM)technique.

关 键 词：阴极荧光 扫描电镜 有机荧光团 亚细胞结构 

分 类 号：Q6[生物学—生物物理学] Q-336