Author Affiliations
Abstract
1 Institute for Quantum Science and Technology, College of Science, National University of Defense Technology, Changsha 410073, China
2 Hunan Key Laboratory of Mechanism and Technology of Quantum Information, Changsha 410073, China
3 School of Physics and Astronomy, University of Glasgow, Glasgow G12 8QQ, UK
The 3D location and dipole orientation of light emitters provide essential information in many biological, chemical, and physical systems. Simultaneous acquisition of both information types typically requires pupil engineering for 3D localization and dual-channel polarization splitting for orientation deduction. Here we report a geometric phase helical point spread function for simultaneously estimating the 3D position and dipole orientation of point emitters. It has a compact and simpler optical configuration compared to polarization-splitting techniques and yields achromatic phase modulation in contrast to pupil engineering based on dynamic phase, showing great potential for single-molecule orientation and localization microscopy.
PSF engineering geometric phase single-molecule orientation and localization microscopy Chinese Optics Letters
2024, 22(3): 031103
国家纳米科学中心中国科学院纳米生物效应与安全性重点实验室，北京 100190
超分辨显微成像技术自诞生以来，凭借其优异的纳米级空间分辨率，已成为生命科学研究中精准揭示复杂生命现象的重要成像技术。其中，基于单分子定位的超分辨成像策略，使得定位、观察、研究单个探针分子独特的理、化、光学性能成为可能。偏振作为荧光信号的一个重要特性，近年来伴随着单分子三维取向成像技术的发展，逐步在单分子成像和超分辨领域中展示出诸多新颖且重要的应用特性。本文总结了单分子三维取向超分辨成像技术的最新进展，介绍并分析了两类主要的单分子三维取向荧光显微技术——基于荧光吸收与辐射偏振调制的单分子三维取向成像方法以及利用点扩散函数工程将单个荧光分子的三维取向信息编码到荧光图像上的成像策略。此外，还探讨了应用于活细胞或单颗粒的其他类型的超分辨取向成像技术。最后，针对单分子三维取向超分辨成像技术发展与应用前景面临的挑战，进行了总结与展望。
显微 单分子荧光 超分辨成像 单分子空间取向 单分子定位显微术 激光与光电子学进展
2024, 61(6): 0618015
Author Affiliations
Abstract
1 Key Laboratory of Biomedical Engineering of Hainan Province, School of Biomedical Engineering, Hainan University, Haikou 570228, P. R. China
2 School of Computer Science and Technology, Hainan University, Haikou 570228, P. R. China
3 Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, P. R. China
Quantitative data analysis in single-molecule localization microscopy (SMLM) is crucial for studying cellular functions at the biomolecular level. In the past decade, several quantitative methods were developed for analyzing SMLM data; however, imaging artifacts in SMLM experiments reduce the accuracy of these methods, and these methods were seldom designed as user-friendly tools. Researchers are now trying to overcome these difficulties by developing easy-to-use SMLM data analysis software for certain image analysis tasks. But, this kind of software did not pay sufficient attention to the impact of imaging artifacts on the analysis accuracy, and usually contained only one type of analysis task. Therefore, users are still facing difficulties when they want to have the combined use of different types of analysis methods according to the characteristics of their data and their own needs. In this paper, we report an ImageJ plug-in called DecodeSTORM, which not only has a simple GUI for human–computer interaction, but also combines artifact correction with several quantitative analysis methods. DecodeSTORM includes format conversion, channel registration, artifact correction (drift correction and localization filtering), quantitative analysis (segmentation and clustering, spatial distribution statistics and colocalization) and visualization. Importantly, these data analysis methods can be combined freely, thus improving the accuracy of quantitative analysis and allowing users to have an optimal combination of methods. We believe DecodeSTORM is a user-friendly and powerful ImageJ plug-in, which provides an easy and accurate data analysis tool for adventurous biologists who are looking for new imaging tools for studying important questions in cell biology.
Journal of Innovative Optical Health Sciences
2023, 16(6): 2350006
哈尔滨工业大学仪器科学与工程学院，黑龙江 哈尔滨 150080
单分子定位技术通过随机激发荧光标记获得一组稀疏的图像序列，同时对荧光点进行亚像素级别定位，最终实现超分辨显微成像。基于拟合的单分子定位算法，如单发射（SE）及多发射（ME）定位算法，通过对估计器性能进行改进提高了单分子定位的精度和速度；然而，受失配误差和串扰误差的影响，SE算法和ME算法在不同密度情况下各有优劣，均无法达到全密度范围内最优的估计效果，并且分别存在荧光分子利用效率低和计算量大的缺点。本文提出了自适应混合发射单分子定位（SM）算法，该算法通过图像荧光发射密度及强度自适应地确定的拟合区域以及所采用的拟合模型及模型初值，有效避免了上述两种误差的影响，达到了全密度范围内一致、良好的定位效果。在仿真和实验数据上将所提SM算法与SE算法、ME算法进行比较，结果显示，SM算法重构图像的分辨率和对比度在不同发射密度下均具有优势。
生物医学 单分子定位显微技术 超分辨成像 单发射模型 多发射模型 自适应算法 中国激光
2023, 50(21): 2107106
1 南开大学弱光非线性光子学教育部重点实验室，物理科学学院，泰达应用物理研究院，天津 300071
2 南开大学细胞应答交叉科学中心，药物化学生物学国家重点实验室，生命科学学院，天津 300071
3 南开大学深圳研究院，广东 深圳 518083
4 山西大学极端光学协同创新中心，太原 山西 030006
膜蛋白在细胞膜上的时空分布形式决定了其活性状态及功能，在调控细胞生命活动过程中起着重要作用。单分子定位超分辨成像（SMLM）技术为在纳米尺度解析膜蛋白的空间分布提供了可能，但分辨率的极大提升对图像准确聚类分割提出了更高要求。基于密度的空间聚类算法（DBSCAN）是常用的聚类方法之一，但其对于膜蛋白分布不均匀的SMLM超分辨图像的分割效果往往不太理想。本文提出了一种结合多次DBSCAN和层次聚类的混合聚类算法，该算法以DBSCAN方法为分割基础，通过进一步的面积阈值分析和层次聚类，在保持超分辨点簇图像精确聚类识别的前提下，仍能保留每个点簇内的多次定位信号。将该算法应用于模拟数据集和实验数据分割得到的轮廓系数等性能普遍优于传统DBSCAN算法。这种混合聚类方法为膜蛋白SMLM超分辨图像的聚类分割提供了新思路和新方法，有助于更精准地分析膜蛋白在纳米尺度上的空间分布信息。
生物光学 单分子定位超分辨成像 超分辨图像分割 膜蛋白 基于密度的空间聚类算法 层次聚类算法
戴太强 1,2,3,4高晔 1,2,3,4马英 5蔡卜磊 1,2,3,4[ ... ]孔亮 1,2,3,4,*
1 军事口腔医学国家重点实验室，陕西 西安 710032
2 国家口腔疾病临床医学研究中心，陕西 西安 710032
3 陕西省口腔疾病临床医学研究中心，陕西 西安 710032
4 第四军医大学口腔医院 颌面外科，陕西 西安 710032
5 西安电子科技大学 物理学院，陕西 西安 710171
观察细胞器间动态相互作用，深入分析作用规律，对于揭示生理病理过程现象背后的机制具有十分重要的意义。传统光学显微镜受到由光波波长和孔径造成的衍射极限的限制，无法观测细胞器纳米级精细结构及细胞器间相互作用的动态变化规律。超分辨显微成像技术的出现为细胞器相互作用研究提供了重要手段，在深入揭示细胞器相互作用规律，阐明生理病理现象深层的机制研究中发挥了重要的作用。文中介绍了受激发射损耗（Stimulated emission depletion, STED）显微成像、结构光照明显微成像（Structured illumination microscopy, SIM）、单分子定位显微成像（Single molecule localization microscopy, SMLM）技术，并总结了这三类超分辨显微成像技术在细胞器相互作用中的应用与现状，为超分辨显微成像技术在细胞器相互作用研究中的应用提供思路拓展。最后，对超分辨显微成像技术在细胞器相互作用研究中的优势与不足进行分析总结，展望了超分辨显微成像技术在活细胞内细胞器相互作用成像中的需求发展趋势，为光学与医学及生物学的交叉融合发展提供一定的参考。
超分辨显微成像 细胞器间相互作用 受激发射损耗显微成像 结构光照明显微成像 单分子定位显微成像 super-resolution microscopy organelle interaction stimulated emission depletion microscopy structured illumination microscopy single molecule localization microscopy 红外与激光工程
2022, 51(11): 20220622
南方科技大学生物医学工程系，广东 深圳 518055
单分子定位显微成像广泛应用于亚细胞结构的三维空间解析。传统的单分子定位分析方法依赖于点扩展函数(PSF)模型拟合，制约了一些较为复杂的PSF模型的应用。深度学习的出现正在逐渐改变单分子定位的分析方法，有望用来分析复杂的PSF模型，并能提供新的方向和思路。介绍了深度学习在单分子定位、稀疏图像重建、PSF设计和高内涵信息提取等方面的应用，并介绍了目前主流的数据模拟方法。最后对深度学习在单分子定位显微成像领域内的发展方向进行了展望。
显微 单分子定位显微镜 深度学习 超分辨 点扩展函数 中国激光
2022, 49(24): 2407206
Author Affiliations
Abstract
1 Department of Hematology, Zhongnan Hospital of Wuhan University, Wuhan 430071, China
2 Key Laboratory of Biomedical Engineering of Hainan Province, School of Biomedical Engineering, Hainan University, Haikou 570228, China
Hematologic malignancies are one of the most common malignant tumors caused by the clonal proliferation and differentiation of hematopoietic and lymphoid stem cells. The examination of bone marrow cells combined with immunodeflciency typing is of great signiflcance to the diagnostic type, treatment and prognosis of hematologic malignancies. Super-resolution fluorescence microscopy (SRM) is a special kind of optical microscopy technology, which breaks the resolution limit and was awarded the Nobel Prize in Chemistry in 2014. With the development of SRM, many related technologies have been applied to the diagnosis and treatment of clinical diseases. It was reported that a major type of SRM technique, single molecule localization microscopy (SMLM), is more sensitive than flow cytometry (FC) in detecting cell membrane antigens' expression, thus enabling better chances in detecting antigens on hematopoietic cells than traditional analytic tools. Furthermore, SRM may be applied to clinical pathology and may guide precision medicine and personalized medicine for clone hematopoietic cell diseases. In this paper, we mainly discuss the application of SRM in clone hematological malignancies.
Hematologic malignancies super-resolution fluorescence microscopy structured illumination microscopy stimulated emission depletion microscopy single molecule localization microscopy Journal of Innovative Optical Health Sciences
2022, 15(2): 2230005
