受激辐射损耗超分辨显微成像系统研究的新进展

王佳林; 严伟; 张佳; 王璐玮; 杨志刚; 屈军乐

doi:10.7498/aps.69.20200168

摘要

由于受到衍射极限的影响, 传统光学显微镜的分辨率被限制在半个波长左右. 近二十年来出现了许多通过不同方法绕过光学衍射极限的超分辨成像技术, 其中, 受激辐射损耗显微(stimulated emission depletion microscopy, STED)通过引入一束环形损耗光来抑制荧光光斑外围荧光分子的发光, 以达到减小点扩散函数的目的, 实现超分辨成像. 经过近些年的发展, STED系统无论从光束的产生、校准和扫描, 还是最后的成像, 都有了很大的发展. 本文将简要介绍STED成像技术的基本原理, 详述STED超分辨成像技术出现至今在光源、扫描及成像系统等方面的进展, 以及在三维成像和多色成像方面的发展现状, STED技术与其他显微技术的结合. 最后, 本文对STED技术近几年的研究新进展进行了系统的论述, 对STED技术未来的发展趋势进行了探讨.

关键词:

Abstract

Due to the influence of the diffraction limit, the lateral spatial resolution and axial spatial resolution of traditional optical microscopes are limited to ~200 nm and ~500 nm, respectively. In the past two decades, with the rapid development of high-intensity lasers, high-sensitivity detectors and other optoelectronic devices, there have been reported many super-resolution imaging techniques that bypass the optical diffraction limit with different methods. Among these techniques, stimulated emission depletion microscopy (STED) technology has the advantages of high imaging resolution and fast imaging speed. This technology uses two lasers for imaging, one of which is used to excite fluorescence, and the other donut-shaped depletion laser is used to suppress the emission of fluorescent molecules around the fluorescent spot, in order to reduce the fluorescence point spread function and achieve super resolution Imaging. After recent years of development, the STED system has got great progress no matter from the generation, calibration and scanning of the beam, and the final imaging. In terms of laser source, new laser sources such as continuous wave beams, supercontinuum laser, stimulated Raman scattered laser, and higher-order Bessel beams have appeared; in scanning and calibration, new efficiency technology such as parallel scanning and automatic calibration have also appeared; In imaging, new methods such as time gating and phasor analysis have emerged to improve imaging quality. These new technologies and methods are of great significance to improve the efficiency of STED system construction and imaging. In addition, this paper also focuses on the ways to expand the imaging functions of the STED system. First, for three-dimensional STED imaging, this paper mainly introduces three methods to realize three-dimensional STED imaging by wavefront non-coherent adjustment, 4Pi and structured light illumination methods. Second, for multi-color imaging, this paper introduces several dual-color and multi-color imaging techniques for special dyes. Third, this paper introduces the combination of STED technology with fluorescence correlation spectroscopy technology, cell expansion technology, scanning ion-conductance microscope, photo-activated localization microscopy/stochastic optical reconstruction microscopy and other technologies. Finally, this paper systematically discusses the new research progress of STED technology in recent years, and discusses the future development trend of STED technology.

Keywords:

stimulated emission depletionmicroscopy /
three-dimensional imaging /
multi-color imaging /
super-resolution

作者及机构信息

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, 深圳　518060

通信作者: 严伟, weiyan@szu.edu.cn ; 屈军乐, jlqu@szu.edu.cn

基金项目: 国际级-国家重点基础研究发展计划(2017YFA0700500)

Authors and contacts

Optoelectronic Device and System, Ministry of Education/Key Laboratory of Guangdong Province, College of Physics and Optoelectronic Engineering, Shenzhen University, Shenzhen 518060, China

Corresponding author: Yan Wei, weiyan@szu.edu.cn ; Qu Jun-Le, jlqu@szu.edu.cn

文章全文 : translate this paragraph

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施引文献

图 1 STED的原理图　(a)能级辐射示意图; (b)光束重合示意图

Fig. 1. The concept of STED: (a)The concept of stimulated emission; (b)the overlapping of beams.

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图 2 STED系统图

Fig. 2. The setup of STED system.

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图 3 基于超连续谱光源的STED系统^[4]

Fig. 3. STED system based on supercontinuum laser source^[4].

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图 4 基于受激拉曼散射光源的STED系统示意图^[5]　(a)基于受激拉曼散射光源的多色STED系统; (b)激光器在光纤中的SRS输出光谱

Fig. 4. Simplified schematic of a STED system with a stimulated Raman-scattering light source^[5]: (a) Multicolor STED system with stimulated Raman-scattering laser source; (b) output spectrum from the SRS fiber.

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图 5 TED光强度的分布曲线^[6]

Fig. 5. The intensity distributions of depletion beam^[6].

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图 6 共聚焦、传统STED与贝塞尔STED成像的分辨率随成像深度的变化曲线^[7]　(a) 40 nm荧光珠在固体琼脂糖样品不同深度成像的分辨率; (b) 40 nm荧光珠在类脑组织灰质不同深度成像的分辨率

Fig. 6. Imaging curves of Confocal、traditional STED and Bessel-STED^[7]: (a) Resolution of 40 nm fluorescent beads at different depths of solid agarose samples; (b) resolution of 40 nm fluorescent beads at different depths of like-gray matter in brain tissue.

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图 7 零阶贝塞尔激发光与高阶贝塞尔STED光^[8]　(a)零阶贝塞尔激发光在XY平面的光强分布; (b)零阶贝塞尔激发光的光强曲线; (c)一阶贝塞尔STED光在XY平面的光强分布; (d)一阶(黑色)与二阶(红色)贝塞尔STED光的光强曲线

Fig. 7. The zero-order Bessel beam for excitation and the higher-order Bessel beam for depletion^[8]: (a) The intensity distribution of zero-order Bessel excitation beam in the XY plane.; (b) the intensity curve of zero-order Bessel excitation beam; (c) the intensity distribution of first-order Bessel depletion beam in the XY plane; (d) the intensity curve of first-order (black) and second-order (red)Bessel depletion beam.

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图 8 STED光束与激发光束的对准结果^[9]　(a)激发光与STED光未对准时的金纳米颗粒图像; (b) (a)中激发光与STED光未对准时所得到的荧光珠的共聚焦(绿色)与STED(红色)图像的合并; (c)沿图(b)中所示虚线的信号强度曲线: 共聚焦(绿色)和STED(红色); (d)激发光与STED光对准情况下的金纳米颗粒图像; (e) (d)中激发光与STED光对准时所得到的荧光珠的共聚焦(绿色)与STED(红色)图像的合并; (f)沿图(e)中所示直线和点划线的信号强度曲线: 共聚焦(绿色)和STED(红色)

Fig. 8. Alignment results of depletion and excitation beams^[9]: (a) Merged result of the excitation focus (green) and a poorly aligned depletion focus(red) using gold nanoparticles; (b) merged result of corresponding confocal (green) and STED(red) images of fluorescent beads imaged with the focus shown in (a); (c) line profiles across the dotted line in (b): confocal (green) and STED (red); (d) merged image of the excitation focus (green) and a well aligned depletion focus (red); (e) merged result of corresponding confocal (green) and STED (red) images of fluorescent beads imaged with the focus shown in (d); (f) line profiles across the solid and dashed lines in (e): confocal (green) and STED (red).

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图 9 双折射光束整形示意图^[10]　(a) easy-STED双折射光束整形装置示意图; (b)光斑重合效果

Fig. 9. Schematic of birefringent beam shaping^[10]: (a) Schematic of an easy-STED microscope using a birefringent beam shaping device; (b) merged image of the excitation beam and a depletion beam.

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图 10 asySLM-STED系统示意图^[11]

Fig. 10. Schematic diagram of easySLM-STED system^[11]

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图 11 并行扫描STED示意图^[17,18]　(a)扩展STED光束对的结果; (b)多点扫描STED光路示意图

Fig. 11. Schematic diagram of parallel scanning STED^[17,18]: (a) Results of extended STED beam pairs; (b) optical path of the multi-point scanning STED.

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图 12 双光子STED系统示意图^[24]

Fig. 12. Schematic diagram of two-photon STED system^[24].

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图 13 双光子STED系统及成像结果^[25]　(a) SW2PE-STED系统示意图; (b) 2PE与2PE-STED成像对比

Fig. 13. Schematic of two-photon STED and imaging results^[25]: (a) Schematic of SW2PE-STED system; (b) 2PE and 2PE-STED imaging.

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图 14 双光源双色STED系统示意图^[28]　(a)双光源双色STED系统示意图; (b) YFP与GFP的荧光发射光谱

Fig. 14. Schematic diagram of two laser source dual-color STED system^[28]: (a) Dual-color STED system with two laser sources; (b) emission spectra of GFP and YFP.

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图 15 基于非相干波前调制的3D-STED成像系统示意图^[30].　插图: 用于实现横向超分辨(HR)或3D超分辨率(3D)的相位板及对应的STED PSF

Fig. 15. Schematic diagram of 3D-STED system with non-coherent wavefront modulation^[30]. Inset: combinations of phase plates and resulting STED PSFs used to achieve either ultimate lateral resolution (HR) or 3D superresolution.

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图 16 利用4π法实现3D-STED成像的系统示意图^[32]

Fig. 16. Schematic diagram of 4π3D-STED system^[32].

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图 17 基于结构光照明的系统图^[35,36]　(a)结构光照明系统图及空间强度分布; (b)结构光照明3D-STED系统示意图

Fig. 17. Structured illumination system^[35,36]: (a) Simplified diagram of the structured illumination apparatus and spatial intensity distribution; (b) structured illumination 3D-STED system.

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图 18 时间门控STED^[41]　(a)快速时间门控STED系统示意图; (b)共聚焦、STED与gate-STED分辨率结果

Fig. 18. Time-gated STED^[41]: (a) Schematic of fast-gated STED system; (b) comparison of spatial resolution for confocal, STED and gate-STED resolution.

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图 19 离线时间门控STED^[44]　(a)共聚焦、STED与off-line gated-STED分辨率结果; (b) T_g、分辨率与PSNR关系曲线

Fig. 19. Off-line gated-STED^[44]: (a) Spatial resolutions of confocal, STED and off-line gated-STED; (b)the relationship between T_g, resolution and PSNR.

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图 20 STED-FLIM成像及相图分析结果^[45]　(a)彩色相位图; (b)荧光珠的共聚焦图像; (c)荧光珠的STED图像(10 mW); (d)时间门STED图像; (e)相图分析后的图像

Fig. 20. STED-FLIM and Phasor-plot analysis^[45]: (a) Phasor color map of the STED-FLIM image; (b) Confocal, (c) STED·(10 mW), (d) time gate STED and (e) Phasor-plot images of fluorescent beads.

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图 21 区域分割法与pSTED-SPLIT方法所得到的荧光珠的成像结果^[47]　(a)区域分割法示意图; (b) pSTED-SPLIT方法示意图; (c)从左到右依次为荧光珠的共聚焦、常规STED、区域分割法STED和pSTED-SPLIT的成像结果

Fig. 21. Imaging results of fluorescent beads using segmentation and pSTED-SPLIT methods^[47]: (a) Schematic diagram of segmentation method; (b) schematic diagram of pSTED-SPLIT method; (c) from left to right: Confocal, conventional STED, segmentation STED, and pSTED-SPLIT imaging results of fluorescence beads.

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图 22 上转换示意图及利用上转换纳米颗粒的成像结果^[48]　(a)上转换能级关系图; (b)上转换纳米颗粒成像结果

Fig. 22. Schematic diagram of upconversion and imaging results^[48]: (a) Diagram of energy levels of upconversion nanoparticles; (b) imaging results of UCNPs.

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图 23 FED成像原理^[53]

Fig. 23. Illustration of FED theory^[53].

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图 24 DE-STED原理图^[55]

Fig. 24. Schematic principle of DE-STED^[55].

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图 25 TIRF-STED-FCS系统示意图^[59]

Fig. 25. Schematic of TIRF-STED-FCS system^[59].

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图 26 脂膜上sSTED-FCS的成像结果^[61]

Fig. 26. sSTED-FCS results of the lipid membrane^[61].

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[2]	Wildanger D, Patton B R, Schill H, et al. 2012 Adv. Mater. 24 309
[3]	Willig K I, Harke B, Medda R, Hell S W 2007 Nat. Methods 4 915 Google Scholar
[4]	Wildanger D, Rittweger E, Kastrup L, Hell S W 2008 Opt. Express 16 9614 Google Scholar
[5]	Rankin B R, Kellner R R, Hell SW 2008 Opt. Lett. 33 2491 Google Scholar
[6]	Török P, Munro P 2004 Opt. Express 12 3605 Google Scholar
[7]	Yu W T, Ji Z H, Dong D S, Yang X S, Xiao Y F, Gong Q H, Xi P, Shi K B 2016 LaserPhotonicsRev. 10 147
[8]	Zhang P, Goodwin P M, Werner J H 2014 Opt. Express 22 12398 Google Scholar
[9]	Gould T J, Kromann E B, Burke D, et al. 2013 Opt. Lett. 38 1860 Google Scholar
[10]	Reuss M, Engelhardt J, Hell S W 2010 Opt. Express 18 1049 Google Scholar
[11]	Görlitz F, Guldbrand S, Runcorn T H, et al. 2018 J. Biophotonics. 11 e201800087 Google Scholar
[12]	Yan L, Gregg P, Karimi E, et al. 2015 Optica 2 900 Google Scholar
[13]	Gael M, Rebecca M, Birka H, Arnold G, Volker W, Hell SW 2010 Opt. Express 18 1302 Google Scholar
[14]	MurJ, Kavčič B, PoberajI 2013 Appl. Opt. 52 6506 Google Scholar
[15]	Wu Y, Wu X D, Toro L, Stefani E 2015 Methods 88 48 Google Scholar
[16]	Wu X D, Toro L, Stefani E, Wu Y 2015 J. Microsc-Oxford 25 731
[17]	Wagner O, Cheshnovsky O, Roichman Y 2013 Novel Techniques in Microscopy Waikoloa Beach, Hawaii, April 14–18, 2013 NM4 B.3
[18]	Bingen P, Reuss M, Engelhardt J, Hell S W 2011 Opt. Express 19 23716 Google Scholar
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受激辐射损耗超分辨显微成像系统研究的新进展