摘要
荧光寿命显微成像技术(fluorescence lifetime imaging microscopy,FLIM)具有特异性强、灵敏度高、可定量测量等优点,被广泛应用于生物医学、材料学等领域的研究.为使FLIM技术更好地适用于高通量数据的快速分析,近年来涌现出多种荧光寿命分析的新算法.其中,相量分析法(phasor analysis,PA)通过将时间域的拟合转化为频率域的直接计算来获得荧光寿命值,与传统的最小二乘拟合法相比,不仅更加简便快速,适用于低光子数情形,而且便于使数据内容可视化和对数据进行聚类分析,因此越来越受到科研人员的青睐.本文详细阐述了相量分析法的基本原理及运用方法,并在此基础上介绍了该方法在细胞代谢状态测量、蛋白质相互作用研究、细胞微环境测量,以及辅助病理诊断和提高超分辨成像分辨率等方面的应用,着重讨论了PA法在这些FLIM应用实例中的优势所在,为相关领域的研究提供有益的参考.最后,对荧光寿命数据的相量分析及其应用的发展方向进行展望.
Fluorescence lifetime imaging microscopy(FLIM) is widely used in biomedical, materials and other fields.It not only has strong specificity and high sensitivity, but also has the capability of quantitative measurement because the fluorescence lifetime is not affected by the intensity of excitation, the concentration of fluorophores and photobleaching, and consequently is able to monitor the changes of microenvironment and reflecting the interaction between molecules. However, its application is limited to some extent by the complexity of data analysis. In order to make FLIM technology more suitable for fast analysis of high-throughput data, a variety of new algorithms for fluorescence lifetime analysis have emerged in recent years, such as phasor analysis,maximum likelihood estimation, first-order moment, Bayesian analysis, and compressed sensing. Among them,the phasor analysis(PA) method obtains the fluorescence lifetime by converting the fitting in the time domain to the direct calculation in the frequency domain. Compared with traditional least-square fitting method, it is not only simpler and faster, but also more suitable for the case of low photon counts. In addition, in the PA approach to FLIM, the fluorescence decay is directly converted into a phasor diagram by simple mathematics,where the phasor points originating from different pixels in the image are represented by the positions in the phasor plot, and thus the graphical representation obtained by PA method is convenient for data visualization and cluster analysis. Therefore, it has become a simple and powerful analysis method for FLIM, and is increasingly favored by researchers. In this paper, the basic principle of PA method and how we can use it are described in detail. And on this basis, the latest application research progress of the method in cell metabolism state measurement, protein interaction study, cell microenvironment measurement, auxiliary pathological diagnosis, and resolution improvement in super-resolution imaging are introduced and summarized. The advantages of PA method in these FLIM applications are focused on, providing useful reference for the research in related fields. Finally, the phasor analysis method for FLIM data analysis and the development trend of its application are prospected.
作者
林丹樱
牛敬敬
刘雄波
张潇
张娇
于斌
屈军乐
Lin Dan-Ying;Niu Jing-Jing;Liu Xiong-Bo;Zhang Xiao;Zhang Jiao;Yu Bin;Qu Jun-Le(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)
出处
《物理学报》
SCIE
EI
CAS
CSCD
北大核心
2020年第16期313-325,共13页
Acta Physica Sinica
基金
国家重点研发计划(批准号:2017YFA0700500)
国家自然科学基金(批准号:61775144,61975131,61620106016,61525503,61835009)
广东省高等学校科技创新(重点)项目(批准号:2016KCXTD007)
广东省自然科学基金(批准号:2018A030313362)
深圳市基础研究项目(批准号:JCYJ20170818144012025,JCYJ20170818141701667,JCYJ20170412105003520)资助的课题。
关键词
荧光寿命显微成像
数据分析
相量分析法
生物医学应用
fluorescence lifetime microscopy
data analysis
phasor analysis
biomedical application
