荧光寿命显微成像技术及应用的最新研究进展

刘雄波; 林丹樱; 吴茜茜; 严伟; 罗腾; 杨志刚; 屈军乐

doi:10.7498/aps.67.20180320

摘要

由于荧光寿命不受探针浓度、激发光强度和光漂白效应等因素影响，荧光寿命显微成像技术（fluorescence lifetime imaging microscopy，FLIM）在监测微环境变化、反映分子间相互作用方面具有高特异性、高灵敏度、可定量测量等优点，近年来已被广泛应用于生物医学等领域.然而，尽管FLIM的发明和发展已历经数十年时间，其在实际应用中仍然面临着许多挑战.例如，其成像分辨率受衍射极限限制，而其成像速度与成像质量和寿命测量精度则存在相互制约的关系.近几年来，相关硬件和软件的快速发展及其与其他光学技术的结合，极大地推动了FLIM技术及其应用的新发展.本文简要介绍了基于时域和频域的不同寿命探测方法的FLIM技术的基本原理及特点，在此基础上概述了该技术的最新研究进展，包括其成像性能的提升和在生物医学应用中的研究现状，详细阐述了近几年来研究者们通过硬件和软件算法的改进以及与自适应光学、超分辨成像技术等新型光学技术的结合来提升FLIM的成像速度、寿命测量精度、成像质量和空间分辨率等方面所做的努力，以及FLIM在生物医学基础研究、疾病诊断与治疗、纳米材料的生物医学研究等方面的应用，最后对其未来发展趋势进行了展望.

关键词:

Abstract

In the past decade, fluorescence lifetime imaging microscopy (FLIM) has been widely used in biomedical research and other fields. As the fluorescence lifetime is unaffected by probe concentration, excitation intensity and photobleaching, the FLIM has the advantages of high specificity, high sensitivity and capability of quantitative measurement in monitoring microenvironment changes and reflecting the intermolecular interactions. Despite decades of technical development, the FLIM technology still faces some challenges in practical applications. For example, its resolution is still difficult to overcome the diffraction limit and the trade-off among imaging speed, image quality and lifetime accuracy needs to be considered. In recent years, a great advance in FLIM and its application has been made due to the rapid development of hardware and software and their integration with other optical technologies. In this review, we first introduce the principle and characteristics of FLIM technology based on time domain and frequency domain. We then summarize the latest progress of FLIM technology:1) imaging speed enhancement based on hardware improvement such as optimized time-correlated single photon counting module, single photon avalanche diode array detector, and acousto-optic deflector scanner; 2) lifetime measurement accuracy improvement by the proposed algorithms such as maximum likelihood estimate, Bayesian analysis and compressed sensing; 3) imaging quality enhancement and spatial resolution improvement by integrating FLIM with other optical technologies such as adaptive optics for correcting the aberration generated in the optical path, special illumination for equipping wide-field FLIM with optical sectioning ability, and super-resolution techniques for exceeding the resolution limit. We then highlight some recent applications in biomedical studies such as signal transduction or plant cell growth, disease diagnosis and treatment in cancers, Alzheimer's disease and skin diseases, assessment for toxicity and treatment efficiency of nanomaterials developed in the past few years. Finally, we present a short discussion on the current challenges and provide an outlook of the future development of enhanced imaging performance for FLIM technology. We hope that our summary on the state-of-the-art FLIM, our commentary on future challenges, and some proposed avenues for further advances will contribute to the development of FLIM technology and its applications in relevant fields.

Keywords:

fluorescence lifetime imaging microscopy /
fast imaging /
super-resolution imaging /
biomedical application

作者及机构信息

1.
深圳大学光电工程学院, 光电子器件与系统教育部/广东省重点实验室, 深圳 518060

通信作者: 林丹樱, dylin@szu.edu.cn;jlqu@szu.edu.cn ; 屈军乐, dylin@szu.edu.cn;jlqu@szu.edu.cn

基金项目: 国家自然科学基金（批准号：61775144，61525503，61620106016，81727804）、国家重点基础研究发展计划（批准号：2015CB352005）和广东省自然科学基金创新研究团队项目（批准号：2014A030312008）资助的课题.

Authors and contacts

1.
Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Optoelectronic Engineering, Shenzhen University, Shenzhen 518060, China

Corresponding author: Lin Dan-Ying, dylin@szu.edu.cn;jlqu@szu.edu.cn ; Qu Jun-Le, dylin@szu.edu.cn;jlqu@szu.edu.cn

Funds: Project supported by the National Natural Science Foundation of China (Grant Nos. 61775144, 61525503, 61620106016, 81727804), the National Basic Research Program of China (Grant No. 2015CB352005), and the Natural Science Foundation of Guangdong Province for Innovation Research Team, China (Grant No. 2014A030312008).

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施引文献

[1]	Lin D Y, Ma W Y 2007 Physics 36 783 (in Chinese)[林丹樱, 马万云 2007 物理 36 783]
[2]	Huang R, Zhou X M 2013 Acta Laser Biol. Sin. 22 289 (in Chinese)[黄茹, 周小明 2013 激光生物学报 22 289]
[3]	Meyer-Almes F J 2017 Methods Appl. Fluores. 5 042002
[4]	Liu C, Zhou Y, Wang X W, Liu Y L 2011 Las. Optoelect. Prog. 48 46 (in Chinese)[刘超, 周燕, 王新伟, 刘育梁 2011 激光与光电子学进展 48 46]
[5]	Becker W 2012 J. Microsc. Oxford 247 119
[6]	Qu J L, Niu H B, Guo B P 1997 Appl. Laser 17 100 (in Chinese)[屈军乐, 牛憨笨, 郭宝平 1997 应用激光 17 100]
[7]	Liu L X, Qu J L, Lin Z Y, Chen D N, Guo B P, Niu H B 2005 J. Shenzhen Univ. Sci. Eng. 22 133 (in Chinese)[刘立新, 屈军乐, 林子扬, 陈丹妮, 郭宝平, 牛憨笨 2005 深圳大学学报(理工版) 22 133]
[8]	Li J 2016 M. S. Thesis (Kaifeng:Henan University) (in Chinese)[李珺 2016 硕士学位论文 (开封:河南大学)]
[9]	Wu Q Q 2017 M. S. Thesis (Shenzhen:Shenzhen University) (in Chinese)[吴茜茜 2017 硕士学位论文 (深圳:深圳大学)]
[10]	Wang X, Uchida T, Minami S 1989 Appl. Spectrosc. 43 840
[11]	Marois A L, Suhling K 2017 Quantitative Live Cell FLIM Imaging in Three Dimensions (Cham, Switzerland:Springer International Publishing) pp31-48
[12]	Alexiev U, Volz P, Boreham A, Brodwolf R 2017 Eur. J. Pharm. Biopharm. 116 111
[13]	Krishnan R V, Saitoh H, Terada H, Centonze V E, Herman B 2003 Rev. Sci. Instrum. 74 2714
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