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Structured illumination super-resolution microscopy technology: review and prospect

Ting-ai CHEN Long-chao CHEN Hui LI Jia YU Yu-feng GAO Wei ZHENG

陈廷爱, 陈龙超, 李慧, 余佳, 高玉峰, 郑炜. 结构光照明超分辨光学显微成像技术与展望[J]. 中国光学, 2018, 11(3): 307-328. doi: 10.3788/CO.20181103.0307
引用本文: 陈廷爱, 陈龙超, 李慧, 余佳, 高玉峰, 郑炜. 结构光照明超分辨光学显微成像技术与展望[J]. 中国光学, 2018, 11(3): 307-328. doi: 10.3788/CO.20181103.0307
CHEN Ting-ai, CHEN Long-chao, LI Hui, YU Jia, GAO Yu-feng, ZHENG Wei. Structured illumination super-resolution microscopy technology: review and prospect[J]. Chinese Optics, 2018, 11(3): 307-328. doi: 10.3788/CO.20181103.0307
Citation: CHEN Ting-ai, CHEN Long-chao, LI Hui, YU Jia, GAO Yu-feng, ZHENG Wei. Structured illumination super-resolution microscopy technology: review and prospect[J]. Chinese Optics, 2018, 11(3): 307-328. doi: 10.3788/CO.20181103.0307

结构光照明超分辨光学显微成像技术与展望

doi: 10.3788/CO.20181103.0307
基金项目: 

国家自然科学基金 No.81701744

中国博士后科学基金资助项目 No.2017M612763

广东省粤港合作项目 No.2014B050505013

深圳市基础研究计划 No.YJ20150521144321005

深圳市基础研究计划 No.JCYJ20150401145529037

深圳市基础研究计划 No.JCYJ20160608214524052

SIAT优秀青年创新基金 No.2016020

详细信息
    作者简介:

    陈廷爱(1988—),男,山西大同人,硕士,研究助理。2014年毕业于北京理工大学,获光学工程硕士学位,主要从事结构光照明显微成像技术方面的研究。E-mail:

    郑炜(1980—),男,浙江绍兴人,博士,研究员,2011年毕业于香港科技大学,获博士学位,主要从事新型光学显微成像技术方面的研究,E-mail:

  • 中图分类号: O436;O438.2

Structured illumination super-resolution microscopy technology: review and prospect

Funds: 

National Natural Science Foundation of China No.81701744

Postdoctoral Science Foundation of China No.2017M612763

Guangdong Province Guangdong-Hongkong Cooperation Project No.2014B050505013

Shenzhen City Basic Research Program No.YJ20150521144321005

Shenzhen City Basic Research Program No.JCYJ20150401145529037

Shenzhen City Basic Research Program No.JCYJ20160608214524052

SIAT Outstanding Youth Innovation Fund No.2016020

More Information
    Author Bio:

    CHEN Ting-ai(1988—), obtained his master′s degree in optical engineering from Beijing Institute of Technology in 2014. His main research interest focuses on structured illumination microscopic imaging technology

    ZHENG Wei(1980—), PhD researcher, graduated from Hong Kong University of Science and Technology with a PhD in 2011. His current research interests are new optical microscopy imaging technology

    Corresponding author: ZHENG Wei, E-mail:zhengwei@siat.ac.cn
  • 摘要: 结构光照明显微镜(Structured Illumination Microscopy,SIM)通过结构化照明在频率域以空间混频的方式将物体高频信息载入光学系统的探测通带内实现突破衍射极限的超分辨光学显微成像。SIM凭借其较低的激发光强、对荧光染料的非特异性需求以及快速的宽场成像优势已成为活细胞超分辨光学显微成像方面应用最多的技术。本文系统回顾了SIM的技术进展,对SIM的基本原理与实现方法进了详细的分析,重点介绍了本课题组研发的基于光谱分辨的单光子激发超分辨显微镜和结合自适应光学的双光子激发超分辨显微镜这两种最新的SIM技术,最后简要讨论了SIM技术在生物成像中的应用及未来发展方向。
  • 图  1  基于相干光照明与非相干光照明的SIM技术。基于相干光照明的SIM技术包括:二维结构光照明显微镜(2D-SIM)与三维结构光照明显微镜(3D-SIM)

    Figure  1.  WF-SIM technologies based on coherent illumination and incoherent illumination. WF-SIM technology based on coherent illumination includes two-dimensional structured illumination microscopy(2D-SIM) and three-dimensional structured illumination microscopy(3D-SIM)

    图  2  非线性SIM技术原理[15]。(a)左图表示随着照明强度增加,激发出的荧光强度逐渐趋于饱和;右图表示不同饱和状态下激发出的荧光信号在时域空间的分布,逐渐表现出高阶谐波信号;(b)下图表示的是不同饱和状态下高阶谐波分量在频域空间的分布,体现更高频分量的出现与增加

    Figure  2.  Principle of nonlinear SIM technology[15]. (a)Left graph shows that the fluorescence intensity tends to be saturated with the increase of the illumination intensity. Right graph shows the distribution of the fluorescence signal at different saturation states in spatial domain, which gradually presents high-order harmonic signals. (b)Figure below shows the distribution of high-order harmonic components at different saturation states in Fourier domain, reflecting the emergence and increase of higher order harmonic components.

    图  3  线性与非线性SIM技术应用于活细胞成像[26]

    Figure  3.  Linear and nonlinear SIM techniques applied to live cell imaging[26]

    图  4  单点扫描结构光照明成像原理与技术。(a)图像扫描显微镜(ISM),(b)光学光子重定位显微镜(OPRA)/二次扫描共聚焦显微镜(RE-scan)

    Figure  4.  Principle rinciple and technology of single PS-SIM. (a)Image scanning microscopy (ISM). (b) Optical photon reassignment microscopy(OPRA)/RE-scan confocal microscopy(RE-scan)

    图  5  多点扫描结构光照明成像原理与技术。(a)多焦点结构光照明显微镜(MSIM); (b)瞬时结构光照明显微镜(iSIM)

    Figure  5.  Principle and technology of Multi-PS-SIM technology. (a)Multifocal structured illumination microscopy(MSIM); (b)Instant structured illumination microscopy(iSIM)

    图  6  基于光谱分辨的单光子激发超分辨显微成像[78]。SYTO 82与LysoTracker Red分别标记了bEnd3型活细胞的细胞核(图中红色)与溶酶体(图中绿色);(a, e)普通的RE-scan超分辨图像;(d, h)基于光谱分辨的RE-scan超分辨图像;(b, f)和(c, g)分别为光谱解混分离出的细胞核和溶酶体;(i)为两种染料的荧光光谱

    Figure  6.  Single-photon excitation superresolution microscopy imaging based on spectral resolution[78]. SYTO 82 and LysoTracker Red respectively label the nuclei(red in the figure) and lysosomes(green in the figure) of bEnd3-type live cells; (a, e) are normal RE-scan super-resolution images; (d, h) are spectrally resolved RE-scan super-resolution images; (b, f) and (c, g) are the nucleus and lysosomes isolated by spectral unmixing; i is the fluorescence spectrum of two dyes

    图  7  结合自适应光学的双光子激发超分辨显微成像[79]。a、b、c、d及e、f分别为普通双光子激发超分辨显微镜、基于自适应光学的双光子激发超分辨显微镜与基于自适应光学的双光子激发超分辨显微镜,并结合图像减卷积处理后的细胞骨架成像结果;g~l分别为e图对应区域的放大图;m表示系统的横向与纵向分辨率;n表示自适应校正前后的波前相位图

    Figure  7.  Two-photon excitation superresolution microscopy combining with adaptive optics[79]. a, b, c, d and e, f are the fluorescence cytoskeleton images taken from two-photon excited super-resolution microscope, two-photon excited super-resolution microscope with adaptive optics and two-photon excited super-resolution microscope with adaptive optics and deconvolution analysis; g-l are respectively enlarged views of corresponding area in figure e; m represents the latral and axial resolutions of the system; n represents the wave front phase diagram before(left)and after(right) the AO correction

    表  1  Implementation methods of WF-SIM technology

    Table  1.   Implementation methods of WF-SIM technology

    WF-SIM Technology 2D-SIM 3D-SIM
    Illumination source Coherent light Incoherent light Coherent light
    Structure light generating device grating/DMD/SLM DMD/SLM grating/SLM
    Probe signal Fluorescence Fluorescence, reflected light Fluorescence
    Nonlinear SIM - -
    下载: 导出CSV

    表  2  Implementation methods of PS-SIM technology

    Table  2.   Implementation methods of PS-SIM technology

    PS-SIM technology ISM OPRA/RE-scan MSIM iSIM
    Excitation mode Single photon/Two photon Single photon/Two photon Single photon/Two photon Single photon
    Scanning device Galvanometer Galvanometer DMD/Spinning disk/lens array+Galvanometer Spinning disk/lens array+Galvanometer
    Photon reassignment mode Digital Optics Digital Optics
    下载: 导出CSV
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  • 收稿日期:  2017-10-13
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Structured illumination super-resolution microscopy technology: review and prospect

doi: 10.3788/CO.20181103.0307
    基金项目:

    国家自然科学基金 No.81701744

    中国博士后科学基金资助项目 No.2017M612763

    广东省粤港合作项目 No.2014B050505013

    深圳市基础研究计划 No.YJ20150521144321005

    深圳市基础研究计划 No.JCYJ20150401145529037

    深圳市基础研究计划 No.JCYJ20160608214524052

    SIAT优秀青年创新基金 No.2016020

    作者简介:

    陈廷爱(1988—),男,山西大同人,硕士,研究助理。2014年毕业于北京理工大学,获光学工程硕士学位,主要从事结构光照明显微成像技术方面的研究。E-mail:

    郑炜(1980—),男,浙江绍兴人,博士,研究员,2011年毕业于香港科技大学,获博士学位,主要从事新型光学显微成像技术方面的研究,E-mail:

    通讯作者: ZHENG Wei, E-mail:zhengwei@siat.ac.cn
  • 中图分类号: O436;O438.2

摘要: 结构光照明显微镜(Structured Illumination Microscopy,SIM)通过结构化照明在频率域以空间混频的方式将物体高频信息载入光学系统的探测通带内实现突破衍射极限的超分辨光学显微成像。SIM凭借其较低的激发光强、对荧光染料的非特异性需求以及快速的宽场成像优势已成为活细胞超分辨光学显微成像方面应用最多的技术。本文系统回顾了SIM的技术进展,对SIM的基本原理与实现方法进了详细的分析,重点介绍了本课题组研发的基于光谱分辨的单光子激发超分辨显微镜和结合自适应光学的双光子激发超分辨显微镜这两种最新的SIM技术,最后简要讨论了SIM技术在生物成像中的应用及未来发展方向。

English Abstract

陈廷爱, 陈龙超, 李慧, 余佳, 高玉峰, 郑炜. 结构光照明超分辨光学显微成像技术与展望[J]. 中国光学, 2018, 11(3): 307-328. doi: 10.3788/CO.20181103.0307
引用本文: 陈廷爱, 陈龙超, 李慧, 余佳, 高玉峰, 郑炜. 结构光照明超分辨光学显微成像技术与展望[J]. 中国光学, 2018, 11(3): 307-328. doi: 10.3788/CO.20181103.0307
CHEN Ting-ai, CHEN Long-chao, LI Hui, YU Jia, GAO Yu-feng, ZHENG Wei. Structured illumination super-resolution microscopy technology: review and prospect[J]. Chinese Optics, 2018, 11(3): 307-328. doi: 10.3788/CO.20181103.0307
Citation: CHEN Ting-ai, CHEN Long-chao, LI Hui, YU Jia, GAO Yu-feng, ZHENG Wei. Structured illumination super-resolution microscopy technology: review and prospect[J]. Chinese Optics, 2018, 11(3): 307-328. doi: 10.3788/CO.20181103.0307
    • Optical microscopy is one of the greatest inventions in human history. Since its “born” in the 17th century, optical microscopy has always played an important role in the study of modern life sciences with its non-destructive and flexible observation methods. However, traditional optical microscopes are limited by Abbe′s diffraction limit[1](about 200 nm), which hinders scientific research on the finer nanoscale science in cells. In order to observe the intracellular molecular structure, localization, and their interactions to further reveal the nature of life phenomena, scientists have successively proposed a series of optical microscopy imaging systems and methods that break the diffraction limit over the past two decades[2-3]. They can be broadly divided into three categories:(1)Stimulated Emission Depletion(STED) based on the reconstruction of point spread function[4-5], which can usually obtain super resolution images with lateral resolution of 20-60 nm, but the loss of optical power up to GW/cm2 restricts its use on living cells. At the same time, limited by the laser output wavelength, only specific fluorescent dyes can be imaged, such as Phototube Fluorescent Proteins; (2)Single Molecule Localization Microscopy(SMLM) based on single molecule localization, including Photo-activated Localization Microscopy(PALM)[6-8] and Stochastic Optical Reconstruction Microscopy(STORM)[9-11]. The image resolution of this technology is usually between 10-30 nm. Although this technology does not require high excitation intensity(kW/cm2), it takes nearly 10 000 exposures to the same sample to acquire a super-resolution image. The lower time resolution also restricts its application in the dynamic observation of living cells. In addition, the requirements for fluorescent dyes are relatively high in this type of technology. The selected fluorescent dye must have ideal “excitation-quenching” efficiency, such as Photoswitchable Fluorescent Proteins; (3)Structured Illumination Microscopy(SIM) based on the expansion of optical transfer function[12-14]. The lateral resolution of super-resolution images is usually between 50-120 nm, and the low excitation intensity(W/cm2), the non-specific requirements for fluorescent dyes, and the advantages of fast wide-field microscopy, make SIM technology the most applicable of these three technologies and SIM is currently the most widely used technique for live-cell super-resolution optical microscopy.

      光学显微镜是人类历史上最伟大的发明之一,自17世纪“诞生”以来,光学显微镜以其无损、灵活的观察手段在现代生命科学的研究中起到了重要作用。然而传统的光学显微镜会受到阿贝光学衍射极限(Abbe diffraction limit)的限制[1](约200 nm),阻碍了科学家对细胞内更细小的纳米尺度的科学研究。为了观察细胞内分子结构、定位以及其相互作用,进一步揭示生命现象的本质,在过去的20年中,科学家们相继提出了一系列突破衍射极限的光学显微成像系统与方法[2-3],大体分为三类:(1)基于点扩散函数改造的受激发射损耗显微技术(Stimulated Emission Depletion, STED)[4-5], 它通常可获得横向分辨率在20~60 nm的超分辨图像,但高达GW/cm2的损耗光功率制约了其在活细胞上的应用,同时受激光器输出波长的限制,只能对特定荧光染料进行成像,例如高光稳定性荧光蛋白(Phtotostabe Fluorescent Proteins);(2)基于单分子定位的超分辨显微成像技术(Single Molecule Localization Microscopy, SMLM),包括光激活定位显微技术(Photoactivated Localization Microscopy, PALM)[6-8]和随机光学重构显微技术(Stochastic Optical Reconstruction Microscopy, STORM)[9-11],该技术的图像分辨率通常在10~30 nm之间,虽然该类技术对激发光强的要求不高(kW/cm2),但是为了采集一幅超分辨图像需要对同一样品进行近万次曝光,较低的时间分辨率同样制约了其在活细胞动态观察中的应用,除此之外,该类技术对荧光染料的要求也比较高,所选的荧光染料必须具有理想的“激发-淬灭”效率,例如光开关荧光蛋白(Photoswitchable Fluorescent Proteins);(3)基于光学传递函数扩展的结构光照明显微技术(Structured Illumination Microscopy, SIM)[12-14],其超分辨图像的横向分辨率通常在50~120 nm之间,较低的激发光强(W/cm2)、对荧光染料的非特异性需求、快速的宽场成像优势,使得SIM技术是这三类技术中最适合,也是目前在活细胞超分辨光学显微成像方面应用最多的技术。

      In this paper, the history of structured illumination microscopy is systematically reviewed, and the commonalities and differences between the basic principles and methods of super-resolution microscopy with wide-field structured illumination and super-resolution microscopy with point-scanning structured illumination are compared in detail. The two latest technologies of single-photon excitation super-resolution microscopy based on spectral resolution and two-photon excitation super-resolution microscopy combined with adaptive optics have been highlighted. Finally, the development prospect of structured illumination super-resolution imaging technology is briefly discussed.

      本文系统回顾了结构光照明显微技术的发展历程,详细对比了宽场结构光照明超分辨显微成像和点扫描结构光照明超分辨显微成像的基本原理与实现方法的共性和差异,重点介绍了本课题组开发的基于光谱分辨的单光子激发超分辨显微镜和结合自适应光学的双光子激发超分辨显微镜两大最新技术,最后,我们简要讨论了结构光照明超分辨成像技术的发展方向。

    • Structural illumination microscopy has been presented to scientists in wide field imaging since its invention. It is also called Wide Field Structured Illumination Microscopy(WF-SIM)[15-16](see Fig. 1). In 1999, prof. Heintzmann first proposed the use of laterally modulated light to illuminate samples, using modulated illumination light to encode high spatial frequency information that cannot be detected by the original objective lens into a detectable low-frequency image. Once the intensity distribution of the modulating illumination light field and the finally detected low-frequency encoded fringes, in which the sample′s high spatial frequency information is superimposed, are known, the original high-frequency information of the sample can be obtained by calculation. They obtained super-resolution images using fluorescent beads in experiments, and called this technique Lateral Modulated Excitation Microscopy(LMEM)[12]. In 2000, Professor Gustafsson proposed the classical Two-dimensional Structured Illumination Microscopy(2D-SIM) on the basis of Heintzmann et al.[13-14], which is also the most familiar structure today. They obtained about two times higher lateral resolution(~120 nm) than the conventional wide-field microscope through experiments, and applied this technique for the first time to fixed biological samples. The structured illumination microscopy at this time can only increase the resolution in the lateral direction but not in the longitudinal direction. In 2001, Frohn et al. extended this technology to three-dimensional space and proposed a theoretical model to improve three-dimensional full spatial resolution by using three-dimensional structured illumination[17]. However, this scheme was not verified by Gustafsson et al. until 2008. This is the unique Three Dimensional Structured Illumination Microscopy(3D-SIM)[18]. Due to the linear fluorescence excitation characteristics, the resolution of either 2D-SIM or 3D-SIM technology can only be increased by two times but not indefinitely. In 2002, Professor Heintzmann introduced non-linear fluorescence excitation technology into structured illumination. Theoretically, the spectrum space of SIM was further expanded and Saturated Patterned Excitation Microscopy(SPEM) based on nonlinear fluorescence excitation was proposed[19]. In 2005, Gustafsson et al. experimentally verified the feasibility of the SPEM technique and obtained imaging results with a lateral resolution of ~40 nm, which is referred to as Saturated Structured Illumination Microscopy(SSIM)[20]. Due to the requirement of higher excitation light intensity(MW/cm2), the early SPEM/SSIM technology has a very strong non-linear photo-bleaching effect, making it difficult to reconstruct ideal super-resolution images and is not suitable for biological samples. In 2008, Hirvonen et al. proposed the theory of non-linear structured illumination based on photoswitchable fluorescent protein[21], which was subsequently verified by Rego et al. in 2012. In this experiment, non-linear fluorescence excitation was achieved with an excitation light intensity reduction of 6 orders of magnitude, and a lateral resolution of ~50 nm was obtained in cell imaging[22]. From 2D to 3D, from linear to nonlinear, after the theory of SIM technology was perfected, scientists committed to apply SIM technology to live cell biological imaging. In 2008, Schermelleh et al. developed the multi-color SIM technology and applied it to the study of nuclear membrane[23]. In 2009, Kner et al. implemented a rapid 2D-SIM imaging technology and applied it to live cell studies. At ~100 nm resolution, 11 super-resolution images per second can be taken[24]. In 2011, Shao et al. used 3D-SIM technology to achieve rapid 3D live-cell imaging[25]. In 2015, Li et al. proposed a novel non-linear SIM technology combined with pattern activation to obtain a spatial resolution of 62 nm in live cell imaging[26].

      图  1  基于相干光照明与非相干光照明的SIM技术。基于相干光照明的SIM技术包括:二维结构光照明显微镜(2D-SIM)与三维结构光照明显微镜(3D-SIM)

      Figure 1.  WF-SIM technologies based on coherent illumination and incoherent illumination. WF-SIM technology based on coherent illumination includes two-dimensional structured illumination microscopy(2D-SIM) and three-dimensional structured illumination microscopy(3D-SIM)

      结构光照明显微术自提出以来主要是以宽场成像的方式展现在科学家们面前的,可以称之为宽场结构光照明显微成像技术(Wide Field Structured Illumination Microscopy, WF-SIM)[15-16](见图 1)。1999年,Heintzmann教授首次提出了这种采用横向调制光照明样品,利用调制照明光将原本物镜探测不到的高空间频率信息编码至可探测到的低频图像中,如果知道调制照明光场的强度分布和最终探测到的叠加了样品高空间频率信息的低频编码条纹后,样品原本的高频信息也就可以通过计算的方式获得。他们利用荧光小球在实验中获得了超分辨率图像,并把这种技术称为横向调制激发显微镜(Laterally Modulated Excitation Microscopy, LMEM)[12]。2000年,Gustafsson教授在Heintzmann等人的基础上提出了经典的二维结构光照明显微成像技术(Two Dimensional Structured Illumination Microscopy, 2D-SIM)[13-14],也是我们现如今最为熟知的结构光照明显微术,通过实验他们获得了较普通宽场显微镜提高约两倍的横向分辨率(~120 nm),并首次将这一技术应用于固定处理的生物样品中。此时的结构光照明显微术只能在横向上提高分辨率,不能在纵向上提高分辨率。2001年,Frohn等人把这一技术拓展到三维空间,提出了利用三维结构光照明提高三维全空间分辨率的理论模型[17],但直至2008年,此方案才由Gustafsson等人实验验证,这便是独特的三维结构光照明显微成像技术(Three Dimensional Structured Illumination Microscopy, 3D-SIM)[18]。受线性荧光激发特性的影响,无论是2D-SIM还是3D-SIM技术的分辨率只能提高两倍,并不能无限提高。2002年,Heintzmann教授将非线性荧光激发技术引入到结构光照明成像中,从理论上进一步扩展SIM的频谱空间,提出了基于非线性荧光激发的饱和结构光激发显微镜(Saturated Patterned Excitation Microscopy, SPEM)[19]。2005年,Gustafsson等人实验验证了SPEM技术的可行性,获得了横向分辨率~40 nm的成像结果,并将之称为饱和结构光照明显微镜(Saturated Structured Illumination Microscopy, SSIM)[20]。由于需要较高的激发光强(MW/cm2),早期的SPEM/SSIM技术存在着非常强的非线性光漂白效应,很难重建出理想的超分辨图像,也不适用于生物样品。2008年,Hirvonen等人提出了基于光开关荧光蛋白实现非线性结构光照明的理论[21],随后于2012年,由Rego等人实验验证,他们在激发光强降低6个数量级的条件下实现了非线性荧光激发,在细胞成像中获得了~50 nm的横向分辨率[22]。由二维到三维,由线性到非线性,在SIM技术的理论被完善后,科学家们致力于将SIM技术应用于活细胞生物成像。2008年,Schermelleh等人开发了多色彩的SIM技术并把它应用于细胞核膜的研究[23]。2009年,Kner等人实现了快速的2D-SIM成像技术并应用于活细胞研究,在~100 nm分辨率的情况下,每秒可拍摄11幅超分辨图像[24]。更进一步,到2011年,Shao等人利用3D-SIM技术实现了快速的三维活细胞成像[25]。2015年,Li等人提出了结合图案激活的新型非线性SIM技术,在活细胞成像中获得了62 nm的空间分辨率[26]

      Any optical system can be regarded as a low-pass filter whose spatial frequency bandwidth(space cutoff frequency:kc=2NA/λ) can be determined by the Optical Transfer Function(OTF). Information above this spatial frequency cannot be passed. The SIM technology encodes high spatial frequency structural information in the sample into a low spatial frequency image by spatial frequency mixing to achieve optical imaging beyond the diffraction limit.

      任何一个光学系统可以看作为一个低通滤波器,其可通过的空间频率带宽(空间截止频率:kc=2NA/λ)由光学传递函数(Optical Transfer Function, OTF)决定,高于这一空间频率的信息都不可以通过。SIM技术通过空间频率混合的方式将样品中高空间频率的结构信息编码至低空间频率的图像来实现超过衍射极限的光学成像。

      WF-SIM technology uses modulated light formed by two or three laser beams interference as sample excitation light(for the sake of simplicity, the following only analyzes the technical principles of 2D-SIM). If the excitation light intensity is weak, due to the linear fluorescence excitation effect, the fluorescence emission intensity is linearly positively correlated with the excitation light intensity, and the fluorescence distribution acquired on the detector image plane can be expressed as[12-13]:

      WF-SIM技术采用两束或三束激光干涉形成的调制光作为样品激发光(为了简单明了,以下仅分析2D-SIM的技术原理)。当激发光强较弱时,由于线性荧光激发效应,荧光发射强度与激发光强度呈线性正相关,探测器像面所采集的荧光分布可表示为[12-13]

      (1)

      where e(r)=1+cos(kc×r+φ) is the excitation light distribution function, s(r) is the sample distribution function labeled with fluorescence, and h(r) is the point spread function(PSF) of the system. In order to analyze the effect of the WF-SIM technology on the spatial frequency expansion of the optical system, the above equation is to be Fourier transformed:

      式中,e(r)=1+cos(kc×r+φ)是激发光分布函数,s(r)是标记了荧光的样品分布函数,h(r)是系统的点扩散函数(point spread function, PSF)。为了分析WF-SIM技术对光学系统空间频率的扩展,需要将上式进行傅里叶变换:

      (2)

      It can be seen from the above equation that by applying structured illumination to the sample plane with a sinusoidal modulation distribution, the WF-SIM technology moves the high spatial frequency information originally blocked by the optical system into the passband detectable by the optical system OTF, which expands the spatial cut-off frequency of the WF-SIM system to kWF_SIM≤2kc.

      可以看出,通过对样品面施以正弦调制分布的结构光照明,WF-SIM技术将原本被光学系统截止掉的高空间频率信息k+ke搬移进光学系统OTF可探测的通带内,使得WF-SIM系统的空间截止频率扩展为kWF_SIM≤2kc

      When the excitation light intensity increases to a certain degree, the fluorescent molecules enter the saturated excitation state, and the fluorescence emission intensity is nonlinearly positively correlated with the excitation light intensity(see Fig. 2). At this time, due to the nonlinear fluorescence excitation effect[19, 22]:

      图  2  非线性SIM技术原理[15]。(a)左图表示随着照明强度增加,激发出的荧光强度逐渐趋于饱和;右图表示不同饱和状态下激发出的荧光信号在时域空间的分布,逐渐表现出高阶谐波信号;(b)下图表示的是不同饱和状态下高阶谐波分量在频域空间的分布,体现更高频分量的出现与增加

      Figure 2.  Principle of nonlinear SIM technology[15]. (a)Left graph shows that the fluorescence intensity tends to be saturated with the increase of the illumination intensity. Right graph shows the distribution of the fluorescence signal at different saturation states in spatial domain, which gradually presents high-order harmonic signals. (b)Figure below shows the distribution of high-order harmonic components at different saturation states in Fourier domain, reflecting the emergence and increase of higher order harmonic components.

      当激发光强增加到一定程度时,荧光分子进入饱和激发状态,荧光发射强度与激发光强度呈非线性正相关(见图 2),此时由于非线性荧光激发效应[19, 22]

      (3)

      where F[e(r)] is the non-linear response of the sample to the structured illumination, which contains the high-order frequencies of the structured illumination. These higher-order frequencies shift the structural information of the higher spatial frequency of the sample to the pass band of the optical system, further expanding the spatial frequency of the WF -SIM.

      式中,F[e(r)]表示样品对照明结构光的非线性响应,它包含了照明结构光的高阶频率,这些高阶频率会将样品更高空间频率的结构信息搬移到光学系统的通带内,进一步拓展WF-SIM的空间频率。

      According to the different lighting sources, structured light generation devices, and detection signals, the WF-SIM technology can be very different when the specific experimental system is built(see Tab. 1). When the illumination light is a coherent laser[13-14, 18, 22-27, 30-34], if incident light is incident on the structured light generating device(e.g., transmission grating, digital micromirror array, phase spatial light modulator), the device of the periodic structure diffracts the incident light into diffracted light including 0 order, ±1 order, ±2 order, etc., and the structured illumination can be formed on the sample plane by mutual interference of diffracted lights of different orders. The 2D-SIM technology obstructs 0-order and other orders, allowing only ±1 orders of two-beam diffracted light to interfere. The 3D-SIM technique is to allow 0-grade, ±1-level three-beam interference(see Fig. 1a). When the illumination light is an incoherent light source such as a high-pressure mercury lamp or LED[28-29], according to object-image conjugate relation and low-pass permeability of the optical system, the conjugate image of the structured light generation device, which includes various frequency informations, may theoretically exist only fundamental and first-order harmonic information after passing through the optical system, and project structured light illumination at the sample plane(see Fig. 1b).

      表 1  WF-SIM技术的实现方法

      Table 1.  Implementation methods of WF-SIM technology

      WF-SIM Technology 2D-SIM 3D-SIM
      Illumination source Coherent light Incoherent light Coherent light
      Structure light generating device grating/DMD/SLM DMD/SLM grating/SLM
      Probe signal Fluorescence Fluorescence, reflected light Fluorescence
      Nonlinear SIM - -

      WF-SIM技术的实现方法根据照明光源、结构光产生装置以及探测信号的不同,在具体实验系统构建时会有很大不同(见表 1)。当照明光为相干性的激光时[13-14, 18, 22-27, 30-34],入射光照射到结构光产生装置(例如,透射光栅、数字微反射镜阵列、相位空间光调制器)上,该周期结构的装置会将入射光衍射成为包含0级、±1级、±2级等的衍射光,借由不同级次衍射光的相互干涉便可在样品面形成结构光照明。2D-SIM技术便是将0级与其他级次遮挡,只让±1级两束衍射光干涉。3D-SIM技术是让0级,±1级三束光干涉(见图 1a)。当照明光为非相干性的光源如高压汞灯或LED时[28-29],根据物像共轭关系与光学系统的低通透过性,包含各种频率信息的结构光产生装置的共轭像在通过光学系统后理论上可以只有基频与一阶谐波信息存在,在样品面投射结构光照明(见图 1b)。

      Through the digital reconstruction of multiple original images, WF-SIM technology can realize super-resolution images. The quality of the reconstructed image may be degraded due to fluctuations in illumination intensity, sample drift, sample bleaching, inaccurate capture of the period and direction of the illumination structure, weak modulation contrast of the illumination structure light, and phase difference jitter between the original images. Fortunately, with the help of a good reconstruction algorithm, the posterior estimates of these influencing factors can be accurately extracted from the original image, and relatively restore the high frequency information in the sample with high-fidelity[29, 35-58]. In recent years, WF-SIM technology has made great progress both in algorithms and hardware. In addition to providing the same multi-color fluorescence excitation function as confocal technology, the imaging speed of 2D-SIM technology has broken through to 79 frames/s. @16.5 μm2[32]. The latest non-linear SIM technology achieves a spatial resolution of 62 nm@100 W/cm2[26](see Fig. 3) with live cell imaging at 20 consecutive time points. In addition, it does not require special sample preparation and can be used with any fluorescent dye. All in all, WF-SIM technology has become the most favorable super-resolution technology for live-cell imaging, and has become more and more popular in the fields of life sciences, biomedicine etc.

      图  3  线性与非线性SIM技术应用于活细胞成像[26]

      Figure 3.  Linear and nonlinear SIM techniques applied to live cell imaging[26]

      WF-SIM技术实现超分辨图像需要通过多幅原始图像的数字重建,重建图像的质量可能因为照明强度的波动、样品漂移、样品漂白、照明结构光周期与方向的不准确捕捉、照明结构光的弱调制对比度、各原始图像之间的相位差抖动等因素变差。幸运的是,借助优良的重建算法可以从原始图像中准确的提取这些影响因素的后验估计值,进而比较真实的还原样品中的高频信息[29, 35-58]。近几年WF-SIM技术无论是在算法还是硬件上都有了长足的进步,除了可以提供与共聚焦技术一样的多色荧光激发功能外,2D-SIM技术的成像速度已突破到了79 frame/s@16.5 μm2[32],最新的非线性SIM技术还在连续20个时间点的活细胞成像中获得了62 nm的空间分辨率@100 W/cm2[26](见图 3),再加上它不需要特殊的样品制备,可以与任何荧光染料一起使用,WF-SIM技术已经成为最适合于活细胞成像的超分辨技术,在生命科学、生物医学等领域已经越来越普及。

    • WF-SIM technology has many advantages. For example, it has no restrictions on fluorescent dyes, and almost all commonly used dyes can be used for imaging, at the same time, it is a wide-field imaging technology that can simultaneously meet large-scale and high-speed imaging requirements, which greatly facilitates biological research. However, wide field imaging also limits its application on thick tissue samples. The power density of the wide-field excitation light is weak, and its excitation light field is susceptible to tissue scattering and cannot penetrate the tissue surface for three-dimensional imaging. So far, these results have been limited to single(layer) cell imaging and it has not been possible to perform super-resolution imaging of thick tissue. In recent years, there have been new changes in structural illumination microscopy, which is based on spatial frequency mixing to achieve an optical transfer function, forming a technique called Point Scanning Structured Illumination Microscopy(PS-SIM)[59](see Fig. 4 and Fig. 5). In 2010, Müller et al. proposed a structured illumination by using an Airy spot focused by an objective lens instead of the grating required for a conventional wide-field SIM, by scanning the Airy disk and recording each of the Airy disk-excited fluorescence images, a super-resolution image was reconstructed. This is called Image Scanning Microscopy(ISM)[60]. However, since this technique requires the collection of 62 500 original images and it takes more than 10 minutes to reconstruct a super-resolution image(10 μm×10 μm), it′s not very practical. In 2012, York et al. used the Digital Micromirror Device(DMD) on the basis of the ISM to simultaneously scan multiple focused Airy disks to increase the imaging speed of the ISM to the second level, and for the first time, the imaging depth of super-resolution technology in biological samples is extended to 45 μm, while the resolution can be maintained at 145 nm. This technique is called Multifocal Structured Illumination Microscopy(MSIM)[61]. Afterwards, under the prompt of MSIM technology, Schulz et al. collected and recorded the images of each rotation angle in a Confocal Spinning-Disk Microscope(CSD), the pixel reassignment operation is performed after the images of all the angles are collected, and the super-resolution imaging is also realized[62]. Both the ISM technology and the MSIM technology need to collect a large number of original images first, and then use post-image reconstruction(pixel reassignment[63]) to generate super-resolution images. At this time, the PS-SIM technology does not have the capability of real-time imaging. In addition, post-image reconstruction will inevitably introduce subjective human factors, and it will also cause artifacts in the final image due to improper image acquisition. In 2013, the De Luca group, the Heintzmann group, and the Shroff group almost simultaneously proposed to perform the shifting and scaling operations required for image reconstruction in optical instead of digital. Specifically, the De Luca team and Prof. Heintzmann introduced optical secondary scanning on the basis of ISM technology to achieve pixel reassignment operations in digital processing. De Luca group called this technique RE-scan confocal microscopy(RE-scan)[64], and Heinzmann group called this technique Optical Photon Reassignment microscopy(OPRA)[65]. The Shroff team combined scanning galvanometers with microlens arrays on the basis of MSIM to realize redistribution of signal photons on the image plane. They named the technology as Instant Structure Illumination Microscopy(iSIM)[66]. With this technology, imaging speeds up to 100 frames/s can be achieved in live cell imaging, thus real-time superresolution imaging(video rate imaging) of biological samples is realized.

      图  4  单点扫描结构光照明成像原理与技术。(a)图像扫描显微镜(ISM),(b)光学光子重定位显微镜(OPRA)/二次扫描共聚焦显微镜(RE-scan)

      Figure 4.  Principle rinciple and technology of single PS-SIM. (a)Image scanning microscopy (ISM). (b) Optical photon reassignment microscopy(OPRA)/RE-scan confocal microscopy(RE-scan)

      图  5  多点扫描结构光照明成像原理与技术。(a)多焦点结构光照明显微镜(MSIM); (b)瞬时结构光照明显微镜(iSIM)

      Figure 5.  Principle and technology of Multi-PS-SIM technology. (a)Multifocal structured illumination microscopy(MSIM); (b)Instant structured illumination microscopy(iSIM)

      WF-SIM技术有不少的优点,包括:它对荧光染料没有任何限制,几乎所有常用的染料都可以用来成像,同时,它是宽场成像技术,可同时满足大范围、高速度成像的需求,这都大大方便了生物学研究。不过也正是因为宽场成像限制了其在厚组织样品上的应用。宽场激发光的功率密度较弱,而且其激发光场易受组织散射的影响,无法穿透组织表面进行三维成像,所以到目前为止,这些成果都局限在单个(层)细胞成像上,还无法对厚组织进行超分辨成像。近几年,基于空间混频实现光学传递函数扩展的结构光照明显微术产生了新的变化,形成了我们称之为点扫描结构光照明显微成像的技术(Point Scanning Structured Illumination Microscopy, PS-SIM)[59](见图 4图 5)。2010年,Müller等人提出一种利用经物镜聚焦形成的艾里斑代替传统宽场SIM所需的光栅来实现结构化照明,通过扫描艾里斑,并记录每个艾里斑激发的荧光图像,进而重建出超分辨图像的技术,他们将这种技术称之为图像扫描显微镜(Image Scanning MicroscopyISM)[60]。但由于该技术需要采集6.25万张原始图像,花费10 min以上,才能重建出一张超分辨图像(10 μm×10 μm),其实用性不是很高。2012年,York等人在ISM的基础上使用数字微反射镜阵列(Digital Micromirror Device, DMD)让多个聚焦艾里斑同时扫描成像,将ISM的成像速度提高到秒级,并首次将超分辨技术在生物样品中的成像深度拓展到45 μm,分辨率还能维持在145 nm。他们将这种技术称之为多焦点结构光照明显微术(Multifocal Structured Illumination Microscopy, MSIM)[61]。而后Schulz等人在MSIM技术的提示下,在转盘式共聚焦显微镜(Confocal Spinning-Disk Microscope, CSD)中,对每个旋转角度的图像进行采集记录,待所有角度的图像都采集完成后进行像素重定位(pixel reassignment)操作,同样实现超分辨成像[62]。无论是ISM技术还是MSIM技术都需要先采集大量的原始图像,再经过后期图像重建(像素重定位[63])来生成超分辨图像,此时的PS-SIM技术并不具备实时成像的潜力,除此之外,后期图像重建势必会引入主观人为因素,也会因为图像采集不当造成最终图像存在伪影。2013年,De Luca小组、Heintzmann小组与Shroff小组几乎同时提出在光学上完成数字图像重建中所需的像素移动和缩放操作。具体实现上,De Luca小组与Heintzmann教授在ISM技术的基础上引入光学二次扫描来实现数字处理中像素重定位的操作,De Luca小组把这种技术称之为二次扫描共聚焦显微镜(RE-scan confocal microscopy, RE-scan)[64],Heintzmann小组把这种技术称之为光学光子重定位显微镜(Optical Photon Reassignment microscopy, OPRA)[65]。Shroff小组则是在MSIM的基础上结合扫描振镜与微透镜阵列实现信号光子在像面上的重新分布,他们将该技术命名为瞬时结构光照明超分辨显微成像技术(Instant Structure Illumination Microscopy, iSIM)[66],利用该技术,他们在活细胞成像中实现最快可达到100 frames/s的成像速度,真正实现了生物样品的实时超分辨成像(视频速度成像)。

      Unlike the WF-SIM technology, which uses modulated light with a specific high spatial frequency, the PS-SIM technology uses an excitation point that contains all spatial frequencies within the cutoff frequency of the optical system as the illumination structured light, and the point spread function of the excitation point is hex(r). Similarly, when there is only a linear fluorescence excitation effect, after one of the fluorescent molecules in the sample is excited, the distribution function of the detector image plane can be expressed as:

      不同于WF-SIM技术中采用特定高空间频率的调制结构光照明,PS-SIM技术则采用包含了光学系统截止频率以内所有空间频率的激发光斑作为照明结构光,激发光斑的点扩散函数为hex(r)。同样地,当只存在线性荧光激发效应时,样品中的一个荧光分子δ(r)被激发后,在探测器像面的分布函数可表示为:

      (4)

      where hem(r) is the point spread function of the emitted fluorescence, d(r) is the pixel distribution function of the detector, and b is the pixel pitch. In fact, due to the optical system magnification, the detector′s pixel size is much smaller than hem(r), i.e.d(r)~δ(r). Perform the Fourier transform on the above formula, we get:

      式中,hem(r)是发射荧光的点扩散函数,d(r)是探测器的像素分布函数,b是像素间距。实际上由于光学系统放大倍率的存在,探测器的像素大小远小于hem(r),即d(r)~δ(r)。对上式进行傅里叶变换:

      (5)

      Based on the spreading effect of the convolutions of the two functions, it can be seen that the spatial frequency of the image formed by the excitation of a fluorescent molecule can be extended to kex+kem, i.e. PS-SIM technology can theoretically extend the spatial frequency of the optical system to kPS-SIM≤2kc. However, if the fluorescence molecular signals corresponding to each scanning laser spot are directly arranged in two-dimensional images, the resolution of the optical system cannot be improved. This is due to the fact that in the PS-SIM technique, a fluorescent molecule δ(r) is collected on the detector by the pixels co-optical axis of the fluorescent molecule δ(r) and the pixels off-optical axis of the fluorescent molecule δ(r). The signal is mainly from the peak signal of hex(r)×[hem(rd(r-n×b)], that is to say, the effective signal collected by the pixel displaced by n×b distances from the fluorescent molecule δ(r) is actually from the pixel displaced by n×b/2 distances from the fluorescent molecule δ(r). Therefore, before arranging the fluorescence molecular signals in a direct sequence into a two-dimensional image, the fluorescence molecular signal must be pixel reassigned. The distribution function of the fluorescent molecules δ(r) after reassignment at the detector plane can be expressed as:

      根据两个函数卷积的展宽效应,可以看出一个荧光分子δ(r)被激发后形成的像的空间频率可扩展为kex+kem,即PS-SIM技术理论上可以将光学系统的空间频率扩展为kPS-SIM≤2kc。但如果将采集到的每一个扫描激光点对应的荧光分子信号按序直接排布成二维图像,并不能提高光学系统分辨率。这是由于在PS-SIM技术中,一个荧光分子δ(r)会被探测器上与荧光分子δ(r)共光轴的像素和与荧光分子δ(r)不共光轴的像素一起采集,不共光轴像素所采集到的最大有效信号主要来自于hex(r)×[hem(rd(r-n×b)]的峰值信号,也就是说,距离荧光分子δ(r)所在光轴n×b处的像素所采集的有效信号实际上来自于距离荧光分子δ(r)所在光轴 处的像素。因此,在对荧光分子信号按序直接排布成二维图像前,须先将荧光分子信号进行像素重定位(pixel reassignment)操作。荧光分子δ(r)在探测器像面重定位后的分布函数可表示为:

      (6)

      Theoretically, PS-SIM technology can also use nonlinear fluorescence excitation effect to achieve higher spatial frequency expansion, but there is no relevant report.

      理论上,PS-SIM技术也可以利用非线性荧光激发效应实现更高空间频率的扩展,但目前并没有文章报道。

      The realization of PS-SIM technology is mainly based on the point scanning illumination microscopy technique, therefore, it can be well compatible with single-photon excitation and two-photon excitation. Depending on the excitation mode, scanning device, and photon reassignment method, PS-SIM technique also has different combinations in hardware system construction(see Tab. 2). In single-photon excitation[60-66], PS-SIM technology has higher light collection efficiency, higher imaging resolution, and better image signal-to-noise ratio than confocal technology. However, since PS-SIM technology relies on the area array detector to collect fluorescence signals, each scanning point signal needs to be detected by each pixel in a spatially split manner. The spit signal itself is very weak, plus the “pixel dwell time” of the spot scanning technology, the camera readout time, and the time of multiple acquisitions of the original image, making the imaging speed of the PS-SIM technology extremely slow, and the initial imaging speed of ISM is only 0.001 6 Hz@10 μm2. In addition, ISM need digital pixel reassignment and deconvolution to achieve superresolution images. The quality of the reconstructed image can be affected by the quality of the reconstruction algorithm and any factors in imaging collecting(see Fig. 4a). In recent years, the optimization of the algorithm and the innovation of hardware technology have greatly improved the imaging speed and imaging quality of PS-SIM[69-77]. Replacing digital pixel reassignment with optical photon reassignment, so-called OPRA/RE-scan, which enables superresolution imaging in one acquisition process, avoiding the time consumption of multiple acquisitions of the original image, but still limited by the “pixel dwell time” of the point scanning technique(see Fig. 4b). Multi-point scanning provides a mode of “parallel excitation” that avoids the “pixel dwell time” limitation of single-point scanning techniques. MSIM scans samples by using sparse two-dimensional illumination patterns generated by high-speed DMDs. Although digital photon reassignment is still used to obtain super-resolution images, the imaging speed is increased to 1 Hz@48 μm2(see Fig. 5a). Further combining multifocal scanning with optical photo reassignment, iSIM increases the imaging speed to 100 Hz and is only limited by the camera read time(see Fig. 5b).

      表 2  PS-SIM技术的实现方法

      Table 2.  Implementation methods of PS-SIM technology

      PS-SIM technology ISM OPRA/RE-scan MSIM iSIM
      Excitation mode Single photon/Two photon Single photon/Two photon Single photon/Two photon Single photon
      Scanning device Galvanometer Galvanometer DMD/Spinning disk/lens array+Galvanometer Spinning disk/lens array+Galvanometer
      Photon reassignment mode Digital Optics Digital Optics

      PS-SIM技术的实现方法主要是基于点扫描成像技术,因此,它可以很好的兼容单光子激发与双光子激发。依据激发模式、扫描装置、光子重定位方式的不同,PS-SIM技术在硬件系统构建上也有着不同的组合(见表 2)。单光子激发时[60-66],PS-SIM技术比共聚焦技术具有更高的光收集效率、更高的成像分辨率及更好的图像信噪比,但由于PS-SIM技术依靠面阵探测器收集荧光信号,每一个扫描点的信号需按空间展开的方式被各像素探测,分散的信号本身就很弱,再加上点扫描技术的“像素停留时间”、相机的读出时间、多次采集原始图像的时间,使得PS-SIM技术的成像速度极其缓慢,最初ISM的成像速度只有0.001 6 Hz@10 μm2。除此外,ISM需要借助数字式的光子重定位与减卷积算法实现超分辨图像,重建算法的优劣与成像过程中的任何因素都会影响重建图像的质量(见图 4a)。近几年减卷积算法的优化与硬件技术的革新使得PS-SIM技术在成像速度与成像质量上都有了极大的提升[69-77]。将数字方式的光子重定位换成光学方式的光子重定位,便是OPRA/RE-scan,它们可在一次采集过程中实现超分辨成像,避免了多次采集原始图像时间上的消耗,但仍然受点扫描技术的“像素停留时间”限制(见图 4b)。多点扫描提供了一种“并行激发”方式,可以避免单点扫描技术的“像素停留时间”限制,MSIM通过采用高速DMD产生的稀疏二维照明图案来扫描样品,虽然仍使用数字式光子重定位获得超分辨图像,却使得成像速度提升到了1 Hz@48 μm2(见图 5a),进一步,将多点扫描与光学方式的光子重定位结合,iSIM将成像速度提升到了100 Hz,仅仅受相机读出时间的限制(见图 5b)。

      Confocal technology has always been the main tool in live-cell imaging, with a relatively high resolution and a relatively good imaging speed. Single-photon excited PS-SIM technology has surpassed confocal technology after combining multifocal scanning with optical photo reassignment. It not only has super-resolution imaging capability, but also has several times greater imaging speed than existing confocal technology. In addition, it remains the multicolor imaging property of the confocal technology. With multicolor super-resolution imaging, biologists can more accurately capture the interactions between different structures at the same location in living cells. There are two main implementations of multi-color imaging. The first is multi-spectral excitation, which performs multiple detections of the fluorescence signal excited by each spectral line and the images were merged to realize multicolor imaging. MSIM and iSIM used this method. However, this technique requires multiple switching of the laser wavelength in the multicolor imaging process, which inevitably causes spatial misalignment in the image, and is not suitable for fast dynamic imaging such as calcium imaging. The second is monochromatic excitation, which adopts multi-channel detection. Fluorescence signals of different wavelengths are separated by a spectroscopic device (such as a grating, a prism, or a filter group), although separate fluorescent signals can be collected from different detection channels. However, due to the wide spectrum of fluorescent proteins and their cross excitation and emission spectra, this technique inevitably causes the spectral crosstalk of the fused image. We have established a spectrally resolved single-photon excitation super-resolution microscope combining the RE-scan technique and the spectral unmixing principle to achieve multi-color superresolution microscopy under single excitation conditions[78](see Fig. 6). It ensures the spectral purity of the multicolor image after fusion. Moreover, since multi-color super-resolution imaging results can be obtained with one imaging, the technique is also very useful in rapid dynamic imaging, such as calcium imaging.

      图  6  基于光谱分辨的单光子激发超分辨显微成像[78]。SYTO 82与LysoTracker Red分别标记了bEnd3型活细胞的细胞核(图中红色)与溶酶体(图中绿色);(a, e)普通的RE-scan超分辨图像;(d, h)基于光谱分辨的RE-scan超分辨图像;(b, f)和(c, g)分别为光谱解混分离出的细胞核和溶酶体;(i)为两种染料的荧光光谱

      Figure 6.  Single-photon excitation superresolution microscopy imaging based on spectral resolution[78]. SYTO 82 and LysoTracker Red respectively label the nuclei(red in the figure) and lysosomes(green in the figure) of bEnd3-type live cells; (a, e) are normal RE-scan super-resolution images; (d, h) are spectrally resolved RE-scan super-resolution images; (b, f) and (c, g) are the nucleus and lysosomes isolated by spectral unmixing; i is the fluorescence spectrum of two dyes

      共聚焦技术之所以一直是活细胞成像中的主力工具,除了比较合适的成像速度外就是相对高的分辨率。单光子激发的PS-SIM技术在结合多点扫描与光学方式的光子重定位后已经完全超越共聚焦技术,不仅具有超分辨成像的能力,而且成像速度是现有共聚焦技术的几倍以上,除此之外还保留了共聚焦技术的多色成像功能。借助多色超分辨成像,生物学家们可以更准确的捕捉到活细胞中同一位置不同结构之间的相互作用。多色成像主要有两种实现方式,第一种是多谱线激发,对每个谱线激发的荧光信号进行多次探测,将图像融合实现多色成像,MSIM与iSIM便是采用这种方式。但是这种技术在多色成像过程中需要多次切换激光波长,会不可避免地造成图像上的空间错位,而且也不适合于钙离子等的快速动态成像。第二种是单色激发,多通道探测的方式。通过分光器件(例如光栅、棱镜或者滤光片组)将不同波长的荧光信号分离,虽然可以从不同的探测通道上收集分离的荧光信号,但由于荧光蛋白宽谱带且相互交叉的激发与发射谱,这种技术不可避免地会造成融合图像的光谱串扰。我们结合RE-scan技术与光谱解混(spectral unmixing)原理建立了一种光谱分辨的单光子激发超分辨显微镜,实现了单次激发条件下的多色超分辨显微成像[78](见图 6),不仅确保了多色融合后图像的光谱纯净性,而且由于我们是一次成像便可获得多色超分辨成像结果,该技术在钙离子等的快速动态成像中有很强的应用价值。

      The greatest advantage of the PS-SIM technology is its combination with two-photon excitation. This technique expands super-resolution technology, which can only observe cell thickness, into super-resolution technology that can observe tissue thickness[67-68, 79]. Whether it is WF-SIM technology, PS-SIM technology based on single photon excitation, or other super-resolution techniques, one of the biggest problems is that the distribution of light field and the intensity of the excitation light are easily impact by the scattering of the tissue. As the depth of imaging increases, the light field gradually deforms and the light intensity gradually decreases. Two-photon excitation uses a longer-wavelength near-infrared laser as the excitation light source, fundamentally reducing the scattering of the excitation light by the tissue. The two-photon excitation-based MSIM and OPRA/RE-scan achieve a lateral resolution of 145 nm and an axial resolution of 400 nm in the living body. The imaging depth exceeds 100 μm and the imaging speed is close to 1 Hz. At present, there is no report on iSIM based on two-photon excitation, but theoretically it can also be as fast as iSIM under single photon excitation, and it can also perform large depth tissue ultra-resolution imaging like the two-photon excitation-based MSIM and OPRA/RE-scan.

      PS-SIM技术被提出后最大的优势便是与双光子激发相结合,将只能观察细胞厚度的超分辨技术拓展为可以观察组织厚度的超分辨技术[67-68, 79]。无论是WF-SIM技术,基于单光子激发的PS-SIM技术,还是其他超分辨技术,一个最大的问题就是激发光的光场分布与光强大小容易受组织散射的影响,随着成像深度的增加,光场逐渐变形,光强逐渐变小。双光子激发使用较长波长的近红外激光作为激发光源,从根本上降低了组织对激发光的散射。基于双光子激发的MSIM与OPRA/RE-scan在活体组织中实现了145 nm的横向分辨率和400 nm的轴向分辨率,成像深度超过100 μm,成像速度接近1 Hz。目前,基于双光子激发的iSIM并没有文章报道,但理论上它也可以像单光子激发下的iSIM一样快速,像基于双光子激发的MSIM与OPRA/RE-scan一样进行大深度的组织超分辨成像。

      Although two-photon excitation does increase the depth of penetration of PS-SIM in super-resolution imaging, as the depth of imaging deepens, the shape of the excitation point will still be distorted, when imaging at large depths, the excited fluorescence signal is also more susceptible to scattering. To solve this problem, adaptive optics based on PS-SIM technology is introduced and a two-photon excitation super-resolution microscope based on adaptive optics[79] is proposed(see Fig. 7). The system combines both super-resolution optical microscopy imaging capability and large-depth 3D imaging capability enabling the penetration depth of super-resolution imaging to increase to 250 μm, while the lateral resolution still maintains at 176 nm, and the longitudinal resolution at 729 nm. Using this technique, high-resolution 3D imaging research is conducted on cells, nematode embryos and larvae, fruit fly slices, and zebrafish embryos, and the imaging results are far superior to conventional two-photon imaging. Because this technique improves photon utilization efficiency and thus reduces the required laser power, it allows developmental biologist to perform high-resolution, three-dimensional, dynamic observations of the development of nematode embryos in up to one hour of continuous three-dimensional imaging.

      图  7  结合自适应光学的双光子激发超分辨显微成像[79]。a、b、c、d及e、f分别为普通双光子激发超分辨显微镜、基于自适应光学的双光子激发超分辨显微镜与基于自适应光学的双光子激发超分辨显微镜,并结合图像减卷积处理后的细胞骨架成像结果;g~l分别为e图对应区域的放大图;m表示系统的横向与纵向分辨率;n表示自适应校正前后的波前相位图

      Figure 7.  Two-photon excitation superresolution microscopy combining with adaptive optics[79]. a, b, c, d and e, f are the fluorescence cytoskeleton images taken from two-photon excited super-resolution microscope, two-photon excited super-resolution microscope with adaptive optics and two-photon excited super-resolution microscope with adaptive optics and deconvolution analysis; g-l are respectively enlarged views of corresponding area in figure e; m represents the latral and axial resolutions of the system; n represents the wave front phase diagram before(left)and after(right) the AO correction

      虽然使用双光子激发确实提高了PS-SIM技术在超分辨成像下的穿透深度,但是随着成像深度的加深,激发光斑的形状仍然会发生变形,相对的,在大深度成像时,激发出来的荧光信号也更容易受散射影响。为了解决这一问题,我们在PS-SIM技术的基础上引入了自适应光学,提出了基于自适应光学的双光子激发超分辨显微镜[79](见图 7)。该系统同时具备超分辨光学显微成像功能和大深度三维成像能力,使光学超分辨成像深度推进至250 μm,横向分辨率依然能保持在176 nm、纵向分辨率保持在729 nm。利用该技术,我们对细胞、线虫胚胎及幼虫、果蝇脑片和斑马鱼胚胎开展了高清晰三维成像研究,成像效果远优于传统双光子成像。由于该技术提高了光子利用效率,从而降低了所需激光功率,可以允许发育学家在长达1个小时的连续三维成像中对线虫胚胎的发育过程开展高清晰的三维动态观测。

    • Confocal microscopy has always been a necessary tool for scientific researchers in the field of life sciences and biomedicine. In recent years, with the improvement of hardware and software in super-resolution technology, structured illumination super-resolution microscopy has become the most favored technology that can completely replace confocal microscopy. At the live-cell imaging level, WF-SIM technology and single-photon excited PS-SIM technology can provide image information far beyond confocal resolution and imaging speed. In vivo imaging, PS-SIM technology combined with two-photon excitation can provide super-resolution image information at large imaging depth, which is not available with traditional confocal technology and two-photon technology. In addition, for specific applications, the SIM technology can also be integrated with other imaging technologies, such as the integration of SIM technology with light sheet illumination technology in embryonic development, the integration of SIM technology with plasma structure in material chemistry.

      无论是在生命科学领域还是生物医学领域,共聚焦显微镜一直是科研工作者的必备工具。近几年,随着超分辨技术在硬件与软件上的完善,结构光照明超分辨显微镜已成为目前最被看好的可以完全取代共聚焦显微镜的技术。活细胞成像层面,WF-SIM技术和单光子激发的PS-SIM技术可以提供远超共聚焦分辨率与成像速度的图像信息。活体成像方面,结合双光子激发的PS-SIM技术则可以提供大成像深度下的超分辨图像信息,这是传统共聚焦技术与双光子技术都不具备的。除此之外,针对特殊的应用,SIM技术还可以与其他成像技术融合,例如针对胚胎发育学,SIM技术与光片照明技术的融合,针对材料化学,SIM技术与等离子体结构的融合。

      After nearly two decades of development, the potential of WF-SIM technology in cell imaging applications has been fully tapped. A researcher with experience in the development of optical instruments can build a set of 2D-SIM or 3D-SIM systems after spending a short period of system construction training and spending less money and effort. A non-optical professional can also use the WF-SIM system to obtain excellent super-resolution images after 1-2 days of sample preparation and system calibration training [57]. In contrast, the PS-SIM technology that has been developed for less than a decade has very large space for development. (1)PS-SIM can not only be well integrated in existing confocal microscopes, but also easy to operate, however, further improvement in resolution is required. One possible implementation method is to combine the photoswitchable fluorescent proteins using nonlinear fluorescence excitation effect. By controling the possibility that the photoswitchable fluorescent proteins is activated with activating light for the first time, and then exciting the excited protein with the excitation light. The resolution of PS-SIM technology can improve again. (2)PS-SIM technology can be perfectly combined with two-photon technology, using strong anti-scattering capability of two-photon in the biological tissue to achieve two-photon super-resolution imaging depth of 250 μm. However, this is still extremely limited compared to the 1 mm imaging depth reported by ordinary two-photon technology. PS-SIM technology can further combine three-photon technology to realize three-photon super-resolution imaging with greater depth of detection. In addition, PS-SIM technology can also be combined with new large-depth imaging technologies such as photoacoustic and OCT to give full play to the advantages of each technology and achieve new breakthroughs in resolution and imaging depth of each technology. (3)Further promote the implementation of iSIM based on two-photon excitation, and further enhance the three-dimensional imaging(volumetric imaging) speed of the microscope under the premise of improving the resolution and detection depth, making this technology play an unprecedented role in the study of neuroscience and immunology.

      经过近20年的发展,WF-SIM技术在细胞成像应用的潜力已经被充分挖掘。一位有光学仪器开发经验的研究人员在经历短时间的系统构建培训后花费较少的财力与精力就可以搭建起一套2D-SIM或者3D-SIM系统。一位非光学专业的研究人员也可以在接受1~2天的样品制备与系统标定培训后使用WF-SIM系统获得优秀的超分辨图像[57]。相比之下,提出至如今不到十年的PS-SIM技术却还有很大的发展空间。(1)PS-SIM不仅可以很好的融合于现有共聚焦显微镜中,而且操作方便,但是需要进一步提高分辨率。一种可能的实现方法是结合光开关荧光蛋白,利用非线性荧光激发效应,通过激活光第一次照射控制光开关荧光蛋白被激活的可能性,再通过激发光激发这些待激发的蛋白就可以实现PS-SIM技术分辨率再提高。(2)PS-SIM技术可以完美的与双光子技术结合,利用双光子在生物组织的强抗散射能力实现250 μm成像深度下的双光子超分辨成像。但这与普通双光子技术报道的1 mm成像深度相比还是极为有限。PS-SIM技术可进一步结合三光子技术实现更大探测深度的三光子超分辨成像。除此之外,PS-SIM技术还可以与光声,OCT等新型大深度成像技术结合,充分发挥各技术的优势,实现各技术分辨率与成像深度的新突破。(3)进一步推进基于双光子激发的iSIM实现,在提升分辨率与探测深度的前提下,进一步提升显微镜的三维成像(体成像)速度,使这一技术在神经科学,免疫学等的研究中发挥前所未有的效用。

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