Structured illumination super-resolution microscopy technology: review and prospect
doi: 10.3788/CO.20181103.0307
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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。Abstract: Structured illumination microscopy(SIM) is capable of providing super-resolution imaging. It breaks the diffraction limit by moving the high-frequency information of objects into the detectable frequency band of the optical imaging system via frequency mixing. Due to its attractive advantages, such as low intensity illumination, independence of particular fluorescent dyes, and rapid wide-field imaging capability, SIM has become the most popular technique for super-resolution imaging of living cells. This paper first systematically summarizes advances in the development of SIM and introduces corresponding principles at the same time. Then, two novel techniques of SIM developed by our group, including the single-photon excited super-resolution microscopy based on spectral unmixing and the two-photon excited super-resolution microscopy combined with adaptive optics, are particularly introduced in detail. At last, the recent applications and future directions of SIM in biological imaging are briefly discussed.
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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 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 √ - - 
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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
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