人红细胞膜骨架超分辨图像的Voronoï分析  

Voronoï Analysis for Super⁃Resolution Image of Human Erythrocyte Membrane Skeleton

作　　者：杨建宇 胡芬 侯梦迪 董浩 陈靖 潘雷霆 Yang Jianyu;Hu Fen;Hou Mengdi;Dong Hao;Chen Jing;Pan Leiting(Key Laboratory of Weak-Light Nonlinear Photonics,Ministry of Education,School of Physics and TEDA Institute of Applied Physics,Nankai University,Tianjin 300071,China;Frontiers Science Center for Cell Responses,State Key Laboratory of Medicinal Chemical Biology,College of Life Sciences,Nankai University,Tianjin 300071,China;Shenzhen Research Institute of Nankai University,Shenzhen 518083,Guangdong,China;Collaborative Innovation Center of Extreme Optics,Shanxi University,Taiyuan 030006,Shanxi,China)

机构地区：[1]弱光非线性光子学教育部重点实验室,南开大学物理科学学院,泰达应用物理研究院,天津300071 [2]药物化学生物学国家重点实验室,南开大学生命科学学院,细胞应答交叉科学中心,天津300071 [3]南开大学深圳研究院,广东深圳518083 [4]极端光学协同创新中心,山西大学,山西太原030006

出　　处：《中国激光》2023年第15期31-40,共10页Chinese Journal of Lasers

基　　金：国家重点研发计划(2022YFC3400600);国家自然科学基金(32227802,12174208);广东省基础与应用基础研究重大项目(2020B0301030009);天津市自然科学基金(20JCYBJC01010);南开大学中央高校基本科研业务费专项资金(2122021337,2122021405);天津市科学技术普及项目(22KPXMRC00230);高等学校学科创新引智计划(B23045)。

摘　　要：成熟人红细胞膜骨架是由膜下多种蛋白组成的三角晶格网状结构,在维持红细胞形态、变形性、运动和代谢等功能方面扮演着重要角色。单分子定位超分辨成像(SMLM)技术在解析骨架超微结构方面展现出了强大的能力,但分辨率的提升对成像分析手段提出了更高要求。作为一种常用的空间分析方法,Voronoï分割在SMLM图像聚类分析中已被广泛应用。笔者利用自主搭建的SMLM超分辨成像系统获得红细胞膜蛋白和骨架蛋白的超分辨点簇图像,对点簇质心进行Voronoï分割,并对Voronoï多边形面积分布进行伽马函数拟合,发现自由膜蛋白CD59的伽马分布峰值对应的x轴坐标x_(peak)为0.78。结合模拟结果,验证了自由膜蛋白CD59呈随机分布。进一步,肌动蛋白、血影蛋白N端和原肌球蛋白的Voronoï分析结果显示它们的x_(peak)均为0.86,而锚蛋白的x_(peak)为0.84,说明骨架膜蛋白呈相对均匀的分布状态,但锚蛋白较其他骨架蛋白更具随机性。Voronoï方法可助力阐释红细胞膜骨架蛋白的空间分布特性,同时也为点簇状SMLM超分辨图像数据的深入提取提供了新思路和新方法。Objective A human mature erythrocyte membrane skeleton is a triangular lattice network composed of various proteins under the membrane,which is essential for the maintenance of cell morphology,deformation,movement,and metabolism.The unique ultrastructural arrangement of the erythrocyte membrane skeleton is fascinating and has attracted many scientists to develop new technologies for imaging and analysis.Emerging single-molecule localization super-resolution microscopy(SMLM)has demonstrated significant capability in resolving the nanoscale ultrastructure of the erythrocyte membrane skeleton;however,the improvement of resolution has put forward high requirements for imaging analysis methods.A Voronoïdiagram is a geometric analysis method that divides points in space into different regions to describe their spatial distribution.It is widely used in space exploration,materials science,machine learning,and other research fields.In recent years,this method has been prominently utilized in SMLM data extraction and analysis,mainly in the clustering and colocalization analysis of“point cluster”-shaped images.Taking advantage of the Voronoïmethod particularly in SMLM image analysis,we aim to apply this method to extract the distribution information of erythrocyte membrane skeleton protein SMLM images,to more quantitatively and accurately reveal skeletal organization characteristics.Methods SMLM super-resolution images of erythrocyte membranes and skeletal proteins were obtained using a self-built SMLM imaging system.Actin was stained with fluorescently labeled phalloidin(Alexa 647-phalloidin).CD59,N terminus ofβ-spectrin,tropomodulin(TMOD),and ankyrin were labeled with specific antibodies.After SMLM imaging,regions of interest in the SMLM images were selected for analysis,and the corresponding point-cloud image was drawn according to the positioning coordinates.The centroid of each point cluster was subsequently acquired using DBCAN clustering analysis,and the image boundary was determined based on the maximum and m

关 键 词：生物光学 超分辨成像 单分子定位 红细胞膜骨架 图像分割 Voronoï分析 

分 类 号：O436[机械工程—光学工程]