抗淬灭增强的ExM‑SOFI技术  

Anti⁃Quenching Enhanced ExM⁃SOFI Technology

作　　者：张丽 刘志佳 费义艳 糜岚 马炯 Zhang Li;Liu Zhijia;Fei Yiyan;Mi Lan;Ma Jiong(The Multiscale Research Institute of Complex Systems(MRICS),School of Life Sciences,Fudan University,Shanghai 200433,China;Department of Optical Science and Engineering,School of Information Science and Engineering,Fudan University,Shanghai 200433,China;Institute of Biomedical Engineering and Technology,Academy for Engineer and Technology,Fudan University,Shanghai 200433,China)

机构地区：[1]复旦大学生命科学学院复杂体系多尺度研究院,上海200433 [2]复旦大学信息科学与工程学院光科学与工程系,上海200433 [3]复旦大学工程技术研究院生物医学工程研究所,上海200433

出　　处：《中国激光》2023年第3期122-129,共8页Chinese Journal of Lasers

基　　金：国家重点研发计划(2021YFF0502900);国家自然科学基金(61575046,11574056)。

摘　　要：膨胀超分辨技术是近几年出现的一种对样品制备进行改进实现分辨率提升的超分辨技术,由于其与其他光学技术的兼容性强,可以进一步提高分辨率,引起了越来越多研究人员的关注。复合膨胀技术是膨胀超分辨技术改进的一个主要发展方向之一,膨胀结合光学波动超分辨技术(ExM‐SOFI)在复合膨胀技术中是一种受限较小且使用较为广泛的技术。为了增强现有ExM‐SOFI技术的成像效果,本课题组将成像缓冲液技术应用于ExM‐SOFI技术,以增强膨胀样品在拍摄过程中的抗淬灭能力,从而使普通染料在ExM‐SOFI中的荧光强度、荧光波动幅度和闪烁比等均有增强。微管和囊泡的染色成像结果表明,使用这种技术可以使样品在高阶SOFI中保持真实结构,伪影更少,因而高阶SOFI技术可以提升膨胀样品的最终分辨率。Objective Expansion superresolution technology,in which resolution is improved by improving the corresponding sample,has emerged in recent years.Owing to its strong compatibility with other optical technologies and its high resolution,it has attracted an increasing amount of research attention.The combination of expansion superresolution technology and other superresolution techniques is one main development direction for expansion superresolution technology.Expansion combined with optical fluctuation superresolution technology(ExMSOFI)is a widely used compound expansion technology with relatively few restrictions.To enhance the imaging of the existing ExMSOFI,we applied an imaging buffer to enhance the antiquenching ability of the expansion sample during shooting.The fluorescence intensity,fluorescence fluctuation amplitude,and ontime ratio of common dyes in ExMSOFI were improved.Finally,the staining results of the microtubules and vesicles indicate that the use of this technique can make the sample more realistic,with fewer artifacts,and can improve the final resolution of expansion samples in highorder SOFI technology.Methods In this study,we derived an antiquenchingenhanced ExMSOFI technique by improving the existing ExMSOFI technique using an imaging buffer.First,the samples were labeled with biotinylated antibodies.Biotins can retain recognition sites after expansion,for postexpansion staining,to reduce signal loss.An expanded hydrogel was then obtained using a common expansion protocol.Next,the expanded hydrogel was cut into a suitable size and reembedded in a highconcentration solution to prevent it from shrinking.After reembedding,the expanded hydrogel was incubated with a dye modified with streptavidin.During photography,the stained hydrogel was immersed in an imaging buffer for imaging.We used an imaging buffer with an oxygenscavenging system as the main component.The fluorescence intensity,antiquenching ability,and fluorescence fluctuation amplitude of the images before and after the buffer treatment

关 键 词：生物光学 ExM‐SOFI 膨胀超分辨 光学波动超分辨 成像缓冲液 抗淬灭增强 

分 类 号：Q63[生物学—生物物理学]