荧光相关光谱技术的研究进展  

New development of fluorescence correlation spectroscopy

作　　者：高欣慰 王璐玮 郭勇 朱殷铷 翁晓羽[1] 严伟[1] 屈军乐[1] Xinwei Gao;Luwei Wang;Yong Guo;Yinru Zhu;Xiaoyu Weng;Wei Yan;Junle Qu(State Key Laboratory of Radio Frequency Heterogeneous Integration(Shenzhen University),Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province,College of Physics and Optoelectronic Engineering,Shenzhen University,Shenzhen 518060,China)

机构地区：[1]深圳大学物理与光电工程学院,光电子器件与系统教育部/广东省重点实验室,射频异质异构集成全国重点实验室(深圳大学),深圳518060

出　　处：《科学通报》2023年第34期4674-4691,共18页Chinese Science Bulletin

基　　金：国家重点研发计划(2021YFF0502900);国家自然科学基金(62127819,62375180,62335008)资助。

摘　　要：荧光相关光谱技术(fluorescence correlation spectroscopy,FCS)将单分子荧光探测技术与统计光谱方法相结合,通过共聚焦荧光测量光学设计与单光子计数方法,测量微小探测体积内由于荧光分子运动所产生的荧光信号涨落,进而对此涨落信号进行关联函数分析,获得分析扩散运动速率、探测空间内平均分子数等重要参数,是研究生物学和生物化学的重要手段.与其他荧光成像和生物物理方法相比,FCS具有高分辨率和高灵敏度等优势,已成为用于量化分子动力学的强大技术,广泛应用于生物医学、生物物理学和化学等领域.本文首先介绍了单点FCS和荧光交叉相关光谱(fluorescence cross-correlation spectroscopy,FCCS)的基本原理,并对FCS和FCCS的实施提供了建议;然后介绍了扫描FCS和图像相关光谱(image correlation spectroscopy,ICS)的基本原理与方法,并对其不同的衍生技术进行了讨论;最后介绍了FCS在膜蛋白、DNA染色体、蛋白质和核酸、酶和多细胞生物及组织中的应用.我们相信,随着技术的发展,FCS有望为生物学研究提供更加准确、细致的信息,并推动人类对复杂生命现象的认知.Fluorescence correlation spectroscopy(FCS)is a powerful technique that combines single-molecule fluorescence detection technology with statistical spectroscopy methods.It utilizes confocal fluorescence measurement optical design and singlephoton counting methods to measure the fluctuation of fluorescence signals caused by the movement of fluorescent molecules in a small detection volume.By analyzing the correlation function of these fluctuations,important parameters such as the rate of diffusion and the average number of molecules in the detection space can be obtained.This technique is widely used in various fields including biomedicine,biophysics,and chemistry due to its high resolution and sensitivity.In 1993,Rigler et al.demonstrated that FCS can be used for single-molecule detection,which opened the doors for the singlepoint FCS application in the biological field.Initially,single-point FCS could only be applied to a single community,but the development of fluorescence cross-correlation spectroscopy(FCCS)made it possible to measure interactions between different species.However,parameters such as labeling efficiency and binding stoichiometry can introduce artifacts in FCCS.Sweeping FCS overcomes the limitation of single-point FCS,which is suitable only for the measurement of slowly diffusing substances or biological structures.Moreover,when combined with stimulated emission depletion(STED)imaging techniques,it enables the study of molecular diffusion patterns at spatial scales below the diffraction limit of light.Bifocal FCS avoids recalibration of the system by defining two overlapping laser spots at fixed distances.Multi-parallel FCS(mp FCS)maximizes data utilization and enables analysis of different FCS techniques in a single measurement.Scanning FCS is widely used for measuring membrane proteins and distinguishing specific protein domains based on different diffusion coefficients.Combined with STED technology,it can directly observe nanoscale dynamics of membrane lipids in living cells.FCS has also been

关 键 词：荧光相关光谱 图像相关光谱 时空图像相关光谱 荧光涨落光谱 并行荧光相关光谱 

分 类 号：O657.3[理学—分析化学]