結(jié)構(gòu)光照明超分辨光學(xué)顯微成像技術(shù)與展望

陳廷愛(ài)，陳龍超，李 慧，余 佳，高玉峰，鄭 煒*

(1.中國(guó)科學(xué)院 深圳先進(jìn)技術(shù)研究院，生物醫(yī)學(xué)光學(xué)與分子影像研究室，廣東 深圳 518055;2.睿芯生命科技(深圳)有限公司，廣東 深圳 518067)

1 Introduction

引 言

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/cm2restricts 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.

光學(xué)顯微鏡是人類歷史上最偉大的發(fā)明之一，自17世紀(jì)“誕生”以來(lái)，光學(xué)顯微鏡以其無(wú)損、靈活的觀察手段在現(xiàn)代生命科學(xué)的研究中起到了重要作用。然而傳統(tǒng)的光學(xué)顯微鏡會(huì)受到阿貝光學(xué)衍射極限(Abbe diffraction limit)的限制[1](約200 nm)，阻礙了科學(xué)家對(duì)細(xì)胞內(nèi)更細(xì)小的納米尺度的科學(xué)研究。為了觀察細(xì)胞內(nèi)分子結(jié)構(gòu)、定位以及其相互作用，進(jìn)一步揭示生命現(xiàn)象的本質(zhì)，在過(guò)去的20年中，科學(xué)家們相繼提出了一系列突破衍射極限的光學(xué)顯微成像系統(tǒng)與方法[2-3]，大體分為三類：(1)基于點(diǎn)擴(kuò)散函數(shù)改造的受激發(fā)射損耗顯微技術(shù)(Stimulated Emission Depletion,STED)[4-5],它通?？色@得橫向分辨率在20～60 nm的超分辨圖像，但高達(dá)GW/cm2的損耗光功率制約了其在活細(xì)胞上的應(yīng)用，同時(shí)受激光器輸出波長(zhǎng)的限制，只能對(duì)特定熒光染料進(jìn)行成像，例如高光穩(wěn)定性熒光蛋白(Phtotostabe Fluorescent Proteins)；(2)基于單分子定位的超分辨顯微成像技術(shù)(Single Molecule Localization Microscopy,SMLM)，包括光激活定位顯微技術(shù)(Photoactivated Localization Microscopy,PALM)[6-8]和隨機(jī)光學(xué)重構(gòu)顯微技術(shù)(Stochastic Optical Reconstruction Microscopy,STORM)[9-11]，該技術(shù)的圖像分辨率通常在10～30 nm之間，雖然該類技術(shù)對(duì)激發(fā)光強(qiáng)的要求不高(kW/cm2)，但是為了采集一幅超分辨圖像需要對(duì)同一樣品進(jìn)行近萬(wàn)次曝光，較低的時(shí)間分辨率同樣制約了其在活細(xì)胞動(dòng)態(tài)觀察中的應(yīng)用，除此之外，該類技術(shù)對(duì)熒光染料的要求也比較高，所選的熒光染料必須具有理想的“激發(fā)-淬滅”效率，例如光開(kāi)關(guān)熒光蛋白(Photoswitchable Fluorescent Proteins)；(3)基于光學(xué)傳遞函數(shù)擴(kuò)展的結(jié)構(gòu)光照明顯微技術(shù)(Structured Illumination Microscopy,SIM)[12-14]，其超分辨圖像的橫向分辨率通常在50～120 nm之間，較低的激發(fā)光強(qiáng)(W/cm2)、對(duì)熒光染料的非特異性需求、快速的寬場(chǎng)成像優(yōu)勢(shì)，使得SIM技術(shù)是這三類技術(shù)中最適合，也是目前在活細(xì)胞超分辨光學(xué)顯微成像方面應(yīng)用最多的技術(shù)。

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.

本文系統(tǒng)回顧了結(jié)構(gòu)光照明顯微技術(shù)的發(fā)展歷程，詳細(xì)對(duì)比了寬場(chǎng)結(jié)構(gòu)光照明超分辨顯微成像和點(diǎn)掃描結(jié)構(gòu)光照明超分辨顯微成像的基本原理與實(shí)現(xiàn)方法的共性和差異，重點(diǎn)介紹了本課題組開(kāi)發(fā)的基于光譜分辨的單光子激發(fā)超分辨顯微鏡和結(jié)合自適應(yīng)光學(xué)的雙光子激發(fā)超分辨顯微鏡兩大最新技術(shù)，最后，我們簡(jiǎn)要討論了結(jié)構(gòu)光照明超分辨成像技術(shù)的發(fā)展方向。

Fig.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) 圖1 基于相干光照明與非相干光照明的SIM技術(shù)?；谙喔晒庹彰鞯腟IM技術(shù)包括：二維結(jié)構(gòu)光照明顯微鏡(2D-SIM)與三維結(jié)構(gòu)光照明顯微鏡(3D-SIM)

2 Wide-field structured illumination microscopic technology

寬場(chǎng)結(jié)構(gòu)光照明顯微成像技術(shù)

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 Heintzmannetal.[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 Gustafssonetal. 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, Gustafssonetal. 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, Hirvonenetal. proposed the theory of non-linear structured illumination based on photoswitchable fluorescent protein[21], which was subsequently verified by Regoetal. 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, Schermellehetal. developed the multi-color SIM technology and applied it to the study of nuclear membrane[23]. In 2009, Kneretal. 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, Shaoetal. used 3D-SIM technology to achieve rapid 3D live-cell imaging[25]. In 2015, Lietal. proposed a novel non-linear SIM technology combined with pattern activation to obtain a spatial resolution of 62 nm in live cell imaging[26].

結(jié)構(gòu)光照明顯微術(shù)自提出以來(lái)主要是以寬場(chǎng)成像的方式展現(xiàn)在科學(xué)家們面前的，可以稱之為寬場(chǎng)結(jié)構(gòu)光照明顯微成像技術(shù)(Wide Field Structured Illumination Microscopy,WF-SIM)[15-16](見(jiàn)圖1)。1999年，Heintzmann教授首次提出了這種采用橫向調(diào)制光照明樣品，利用調(diào)制照明光將原本物鏡探測(cè)不到的高空間頻率信息編碼至可探測(cè)到的低頻圖像中，如果知道調(diào)制照明光場(chǎng)的強(qiáng)度分布和最終探測(cè)到的疊加了樣品高空間頻率信息的低頻編碼條紋后，樣品原本的高頻信息也就可以通過(guò)計(jì)算的方式獲得。他們利用熒光小球在實(shí)驗(yàn)中獲得了超分辨率圖像，并把這種技術(shù)稱為橫向調(diào)制激發(fā)顯微鏡(Laterally Modulated Excitation Microscopy,LMEM)[12]。2000年，Gustafsson教授在Heintzmann等人的基礎(chǔ)上提出了經(jīng)典的二維結(jié)構(gòu)光照明顯微成像技術(shù)(Two Dimensional Structured Illumination Microscopy,2D-SIM)[13-14]，也是我們現(xiàn)如今最為熟知的結(jié)構(gòu)光照明顯微術(shù)，通過(guò)實(shí)驗(yàn)他們獲得了較普通寬場(chǎng)顯微鏡提高約兩倍的橫向分辨率(～120 nm)，并首次將這一技術(shù)應(yīng)用于固定處理的生物樣品中。此時(shí)的結(jié)構(gòu)光照明顯微術(shù)只能在橫向上提高分辨率，不能在縱向上提高分辨率。2001年，F(xiàn)rohn等人把這一技術(shù)拓展到三維空間，提出了利用三維結(jié)構(gòu)光照明提高三維全空間分辨率的理論模型[17]，但直至2008年，此方案才由Gustafsson等人實(shí)驗(yàn)驗(yàn)證，這便是獨(dú)特的三維結(jié)構(gòu)光照明顯微成像技術(shù)(Three Dimensional Structured Illumination Microscopy,3D-SIM)[18]。受線性熒光激發(fā)特性的影響，無(wú)論是2D-SIM還是3D-SIM技術(shù)的分辨率只能提高兩倍，并不能無(wú)限提高。2002年，Heintzmann教授將非線性熒光激發(fā)技術(shù)引入到結(jié)構(gòu)光照明成像中，從理論上進(jìn)一步擴(kuò)展SIM的頻譜空間，提出了基于非線性熒光激發(fā)的飽和結(jié)構(gòu)光激發(fā)顯微鏡(Saturated Patterned Excitation Microscopy,SPEM)[19]。2005年，Gustafsson等人實(shí)驗(yàn)驗(yàn)證了SPEM技術(shù)的可行性，獲得了橫向分辨率～40 nm的成像結(jié)果，并將之稱為飽和結(jié)構(gòu)光照明顯微鏡(Saturated Structured Illumination Microscopy,SSIM)[20]。由于需要較高的激發(fā)光強(qiáng)(MW/cm2)，早期的SPEM/SSIM技術(shù)存在著非常強(qiáng)的非線性光漂白效應(yīng)，很難重建出理想的超分辨圖像，也不適用于生物樣品。2008年，Hirvonen等人提出了基于光開(kāi)關(guān)熒光蛋白實(shí)現(xiàn)非線性結(jié)構(gòu)光照明的理論[21]，隨后于2012年，由Rego等人實(shí)驗(yàn)驗(yàn)證，他們?cè)诩ぐl(fā)光強(qiáng)降低6個(gè)數(shù)量級(jí)的條件下實(shí)現(xiàn)了非線性熒光激發(fā)，在細(xì)胞成像中獲得了～50 nm的橫向分辨率[22]。由二維到三維，由線性到非線性，在SIM技術(shù)的理論被完善后，科學(xué)家們致力于將SIM技術(shù)應(yīng)用于活細(xì)胞生物成像。2008年，Schermelleh等人開(kāi)發(fā)了多色彩的SIM技術(shù)并把它應(yīng)用于細(xì)胞核膜的研究[23]。2009年，Kner等人實(shí)現(xiàn)了快速的2D-SIM成像技術(shù)并應(yīng)用于活細(xì)胞研究，在～100 nm分辨率的情況下，每秒可拍攝11幅超分辨圖像[24]。更進(jìn)一步，到2011年，Shao等人利用3D-SIM技術(shù)實(shí)現(xiàn)了快速的三維活細(xì)胞成像[25]。2015年，Li等人提出了結(jié)合圖案激活的新型非線性SIM技術(shù)，在活細(xì)胞成像中獲得了62 nm的空間分辨率[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.

任何一個(gè)光學(xué)系統(tǒng)可以看作為一個(gè)低通濾波器，其可通過(guò)的空間頻率帶寬(空間截止頻率：kc=2NA/λ)由光學(xué)傳遞函數(shù)(Optical Transfer Function,OTF)決定，高于這一空間頻率的信息都不可以通過(guò)。SIM技術(shù)通過(guò)空間頻率混合的方式將樣品中高空間頻率的結(jié)構(gòu)信息編碼至低空間頻率的圖像來(lái)實(shí)現(xiàn)超過(guò)衍射極限的光學(xué)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技術(shù)采用兩束或三束激光干涉形成的調(diào)制光作為樣品激發(fā)光(為了簡(jiǎn)單明了，以下僅分析2D-SIM的技術(shù)原理)。當(dāng)激發(fā)光強(qiáng)較弱時(shí)，由于線性熒光激發(fā)效應(yīng)，熒光發(fā)射強(qiáng)度與激發(fā)光強(qiáng)度呈線性正相關(guān)，探測(cè)器像面所采集的熒光分布可表示為[12-13]：

i(r)=[s(r)×e(r)]?h(r) ,

(1)

wheree(r)=1+cos(kc×r+φ) is the excitation light distribution function,s(r) is the sample distribution function labeled with fluorescence, andh(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)是激發(fā)光分布函數(shù)，s(r)是標(biāo)記了熒光的樣品分布函數(shù)，h(r)是系統(tǒng)的點(diǎn)擴(kuò)散函數(shù)(point spread function,PSF)。為了分析WF-SIM技術(shù)對(duì)光學(xué)系統(tǒng)空間頻率的擴(kuò)展，需要將上式進(jìn)行傅里葉變換：

I(k)=[S(k)?E(k)]×H(k)]=

(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 tokWF_SIM≤2kc.

可以看出，通過(guò)對(duì)樣品面施以正弦調(diào)制分布的結(jié)構(gòu)光照明，WF-SIM技術(shù)將原本被光學(xué)系統(tǒng)截止掉的高空間頻率信息k+ke搬移進(jìn)光學(xué)系統(tǒng)OTF可探測(cè)的通帶內(nèi)，使得WF-SIM系統(tǒng)的空間截止頻率擴(kuò)展為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]:

當(dāng)激發(fā)光強(qiáng)增加到一定程度時(shí)，熒光分子進(jìn)入飽和激發(fā)狀態(tài)，熒光發(fā)射強(qiáng)度與激發(fā)光強(qiáng)度呈非線性正相關(guān)(見(jiàn)圖2)，此時(shí)由于非線性熒光激發(fā)效應(yīng)[19,22]：

s(r)×F[e(r)]=s(r)×[a0+

a1e(r)+a2e(r)2+…+ane(r)n] ,

(3)

whereF[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(xiàn)[e(r)]表示樣品對(duì)照明結(jié)構(gòu)光的非線性響應(yīng)，它包含了照明結(jié)構(gòu)光的高階頻率，這些高階頻率會(huì)將樣品更高空間頻率的結(jié)構(gòu)信息搬移到光學(xué)系統(tǒng)的通帶內(nèi)，進(jìn)一步拓展WF-SIM的空間頻率。

Fig.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. 圖2 非線性SIM技術(shù)原理[15]。(a)左圖表示隨著照明強(qiáng)度增加，激發(fā)出的熒光強(qiáng)度逐漸趨于飽和；右圖表示不同飽和狀態(tài)下激發(fā)出的熒光信號(hào)在時(shí)域空間的分布，逐漸表現(xiàn)出高階諧波信號(hào)；(b)下圖表示的是不同飽和狀態(tài)下高階諧波分量在頻域空間的分布，體現(xiàn)更高頻分量的出現(xiàn)與增加

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).

WF-SIM技術(shù)的實(shí)現(xiàn)方法根據(jù)照明光源、結(jié)構(gòu)光產(chǎn)生裝置以及探測(cè)信號(hào)的不同，在具體實(shí)驗(yàn)系統(tǒng)構(gòu)建時(shí)會(huì)有很大不同(見(jiàn)表1)。當(dāng)照明光為相干性的激光時(shí)[13-14,18,22-27,30-34]，入射光照射到結(jié)構(gòu)光產(chǎn)生裝置(例如，透射光柵、數(shù)字微反射鏡陣列、相位空間光調(diào)制器)上，該周期結(jié)構(gòu)的裝置會(huì)將入射光衍射成為包含0級(jí)、±1級(jí)、±2級(jí)等的衍射光，借由不同級(jí)次衍射光的相互干涉便可在樣品面形成結(jié)構(gòu)光照明。2D-SIM技術(shù)便是將0級(jí)與其他級(jí)次遮擋，只讓±1級(jí)兩束衍射光干涉。3D-SIM技術(shù)是讓0級(jí)，±1級(jí)三束光干涉(見(jiàn)圖1a)。當(dāng)照明光為非相干性的光源如高壓汞燈或LED時(shí)[28-29]，根據(jù)物像共軛關(guān)系與光學(xué)系統(tǒng)的低通透過(guò)性，包含各種頻率信息的結(jié)構(gòu)光產(chǎn)生裝置的共軛像在通過(guò)光學(xué)系統(tǒng)后理論上可以只有基頻與一階諧波信息存在，在樣品面投射結(jié)構(gòu)光照明(見(jiàn)圖1b)。

Tab.1 Implementation methods of WF-SIM technology

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, biomedicineetc.

WF-SIM技術(shù)實(shí)現(xiàn)超分辨圖像需要通過(guò)多幅原始圖像的數(shù)字重建，重建圖像的質(zhì)量可能因?yàn)檎彰鲝?qiáng)度的波動(dòng)、樣品漂移、樣品漂白、照明結(jié)構(gòu)光周期與方向的不準(zhǔn)確捕捉、照明結(jié)構(gòu)光的弱調(diào)制對(duì)比度、各原始圖像之間的相位差抖動(dòng)等因素變差。幸運(yùn)的是，借助優(yōu)良的重建算法可以從原始圖像中準(zhǔn)確的提取這些影響因素的后驗(yàn)估計(jì)值，進(jìn)而比較真實(shí)的還原樣品中的高頻信息[29,35-58]。近幾年WF-SIM技術(shù)無(wú)論是在算法還是硬件上都有了長(zhǎng)足的進(jìn)步，除了可以提供與共聚焦技術(shù)一樣的多色熒光激發(fā)功能外，2D-SIM技術(shù)的成像速度已突破到了79 frame/s@16.5 μm2[32]，最新的非線性SIM技術(shù)還在連續(xù)20個(gè)時(shí)間點(diǎn)的活細(xì)胞成像中獲得了62 nm的空間分辨率@100 W/cm2[26](見(jiàn)圖3)，再加上它不需要特殊的樣品制備，可以與任何熒光染料一起使用，WF-SIM技術(shù)已經(jīng)成為最適合于活細(xì)胞成像的超分辨技術(shù)，在生命科學(xué)、生物醫(yī)學(xué)等領(lǐng)域已經(jīng)越來(lái)越普及。

Fig.3 Linear and nonlinear SIM techniques applied to live cell imaging[26] 圖3 線性與非線性SIM技術(shù)應(yīng)用于活細(xì)胞成像[26]

3 Point scanning structured illumination microscopy imaging technology

點(diǎn)掃描結(jié)構(gòu)光照明顯微成像技術(shù)

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, Mülleretal. 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, Yorketal. 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, Schulzetal. 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.

Fig.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) 圖4 單點(diǎn)掃描結(jié)構(gòu)光照明成像原理與技術(shù)。(a)圖像掃描顯微鏡(ISM)，(b)光學(xué)光子重定位顯微鏡(OPRA)/二次掃描共聚焦顯微鏡(RE-scan)

Fig.5 Principle and technology of Multi-PS-SIM technology. (a)Multifocal structured illumination microscopy(MSIM); (b)Instant structured illumination microscopy(iSIM) 圖5 多點(diǎn)掃描結(jié)構(gòu)光照明成像原理與技術(shù)。(a)多焦點(diǎn)結(jié)構(gòu)光照明顯微鏡(MSIM); (b)瞬時(shí)結(jié)構(gòu)光照明顯微鏡(iSIM)

WF-SIM技術(shù)有不少的優(yōu)點(diǎn)，包括：它對(duì)熒光染料沒(méi)有任何限制，幾乎所有常用的染料都可以用來(lái)成像，同時(shí)，它是寬場(chǎng)成像技術(shù)，可同時(shí)滿足大范圍、高速度成像的需求，這都大大方便了生物學(xué)研究。不過(guò)也正是因?yàn)閷拡?chǎng)成像限制了其在厚組織樣品上的應(yīng)用。寬場(chǎng)激發(fā)光的功率密度較弱，而且其激發(fā)光場(chǎng)易受組織散射的影響，無(wú)法穿透組織表面進(jìn)行三維成像，所以到目前為止，這些成果都局限在單個(gè)(層)細(xì)胞成像上，還無(wú)法對(duì)厚組織進(jìn)行超分辨成像。近幾年，基于空間混頻實(shí)現(xiàn)光學(xué)傳遞函數(shù)擴(kuò)展的結(jié)構(gòu)光照明顯微術(shù)產(chǎn)生了新的變化，形成了我們稱之為點(diǎn)掃描結(jié)構(gòu)光照明顯微成像的技術(shù)(Point Scanning Structured Illumination Microscopy,PS-SIM)[59](見(jiàn)圖4和圖5)。2010年，Müller等人提出一種利用經(jīng)物鏡聚焦形成的艾里斑代替?zhèn)鹘y(tǒng)寬場(chǎng)SIM所需的光柵來(lái)實(shí)現(xiàn)結(jié)構(gòu)化照明，通過(guò)掃描艾里斑，并記錄每個(gè)艾里斑激發(fā)的熒光圖像，進(jìn)而重建出超分辨圖像的技術(shù)，他們將這種技術(shù)稱之為圖像掃描顯微鏡(Image Scanning MicroscopyISM)[60]。但由于該技術(shù)需要采集6.25萬(wàn)張?jiān)紙D像，花費(fèi)10 min以上，才能重建出一張超分辨圖像(10 μm×10 μm)，其實(shí)用性不是很高。2012年，York等人在ISM的基礎(chǔ)上使用數(shù)字微反射鏡陣列(Digital Micromirror Device,DMD)讓多個(gè)聚焦艾里斑同時(shí)掃描成像，將ISM的成像速度提高到秒級(jí)，并首次將超分辨技術(shù)在生物樣品中的成像深度拓展到45 μm，分辨率還能維持在145 nm。他們將這種技術(shù)稱之為多焦點(diǎn)結(jié)構(gòu)光照明顯微術(shù)(Multifocal Structured Illumination Microscopy,MSIM)[61]。而后Schulz等人在MSIM技術(shù)的提示下，在轉(zhuǎn)盤(pán)式共聚焦顯微鏡(Confocal Spinning-Disk Microscope,CSD)中，對(duì)每個(gè)旋轉(zhuǎn)角度的圖像進(jìn)行采集記錄，待所有角度的圖像都采集完成后進(jìn)行像素重定位(pixel reassignment)操作，同樣實(shí)現(xiàn)超分辨成像[62]。無(wú)論是ISM技術(shù)還是MSIM技術(shù)都需要先采集大量的原始圖像，再經(jīng)過(guò)后期圖像重建(像素重定位[63])來(lái)生成超分辨圖像，此時(shí)的PS-SIM技術(shù)并不具備實(shí)時(shí)成像的潛力，除此之外，后期圖像重建勢(shì)必會(huì)引入主觀人為因素，也會(huì)因?yàn)閳D像采集不當(dāng)造成最終圖像存在偽影。2013年，De Luca小組、Heintzmann小組與Shroff小組幾乎同時(shí)提出在光學(xué)上完成數(shù)字圖像重建中所需的像素移動(dòng)和縮放操作。具體實(shí)現(xiàn)上，De Luca小組與Heintzmann教授在ISM技術(shù)的基礎(chǔ)上引入光學(xué)二次掃描來(lái)實(shí)現(xiàn)數(shù)字處理中像素重定位的操作，De Luca小組把這種技術(shù)稱之為二次掃描共聚焦顯微鏡(RE-scan confocal microscopy,RE-scan)[64]，Heintzmann小組把這種技術(shù)稱之為光學(xué)光子重定位顯微鏡(Optical Photon Reassignment microscopy,OPRA)[65]。Shroff小組則是在MSIM的基礎(chǔ)上結(jié)合掃描振鏡與微透鏡陣列實(shí)現(xiàn)信號(hào)光子在像面上的重新分布，他們將該技術(shù)命名為瞬時(shí)結(jié)構(gòu)光照明超分辨顯微成像技術(shù)(Instant Structure Illumination Microscopy,iSIM)[66]，利用該技術(shù)，他們?cè)诨罴?xì)胞成像中實(shí)現(xiàn)最快可達(dá)到100 frames/s的成像速度，真正實(shí)現(xiàn)了生物樣品的實(shí)時(shí)超分辨成像(視頻速度成像)。

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 ishex(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技術(shù)中采用特定高空間頻率的調(diào)制結(jié)構(gòu)光照明，PS-SIM技術(shù)則采用包含了光學(xué)系統(tǒng)截止頻率以內(nèi)所有空間頻率的激發(fā)光斑作為照明結(jié)構(gòu)光，激發(fā)光斑的點(diǎn)擴(kuò)散函數(shù)為hex(r)。同樣地，當(dāng)只存在線性熒光激發(fā)效應(yīng)時(shí)，樣品中的一個(gè)熒光分子δ(r)被激發(fā)后，在探測(cè)器像面的分布函數(shù)可表示為：

?δ(r) ,

(4)

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

式中，hem(r)是發(fā)射熒光的點(diǎn)擴(kuò)散函數(shù)，d(r)是探測(cè)器的像素分布函數(shù)，b是像素間距。實(shí)際上由于光學(xué)系統(tǒng)放大倍率的存在，探測(cè)器的像素大小遠(yuǎn)小于hem(r)，即d(r)～δ(r)。對(duì)上式進(jìn)行傅里葉變換：

?[Hem(k)×exp-i2π(k×nb)]} ,

(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 tokex+kem, i.e. PS-SIM technology can theoretically extend the spatial frequency of the optical system tokPS-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 moleculeδ(r) is collected on the detector by the pixels co-optical axis of the fluorescent moleculeδ(r) and the pixels off-optical axis of the fluorescent moleculeδ(r). The signal is mainly from the peak signal ofhex(r)×[hem(r)×d(r-n×b)], that is to say, the effective signal collected by the pixel displaced byn×bdistances from the fluorescent moleculeδ(r) is actually from the pixel displaced byn×b/2 distances from the fluorescent moleculeδ(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 moleculesδ(r) after reassignment at the detector plane can be expressed as:

?δ(r) .

(6)

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

理論上，PS-SIM技術(shù)也可以利用非線性熒光激發(fā)效應(yīng)實(shí)現(xiàn)更高空間頻率的擴(kuò)展，但目前并沒(méi)有文章報(bào)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).

PS-SIM技術(shù)的實(shí)現(xiàn)方法主要是基于點(diǎn)掃描成像技術(shù)，因此，它可以很好的兼容單光子激發(fā)與雙光子激發(fā)。依據(jù)激發(fā)模式、掃描裝置、光子重定位方式的不同，PS-SIM技術(shù)在硬件系統(tǒng)構(gòu)建上也有著不同的組合(見(jiàn)表2)。單光子激發(fā)時(shí)[60-66]，PS-SIM技術(shù)比共聚焦技術(shù)具有更高的光收集效率、更高的成像分辨率及更好的圖像信噪比，但由于PS-SIM技術(shù)依靠面陣探測(cè)器收集熒光信號(hào)，每一個(gè)掃描點(diǎn)的信號(hào)需按空間展開(kāi)的方式被各像素探測(cè)，分散的信號(hào)本身就很弱，再加上點(diǎn)掃描技術(shù)的“像素停留時(shí)間”、相機(jī)的讀出時(shí)間、多次采集原始圖像的時(shí)間，使得PS-SIM技術(shù)的成像速度極其緩慢，最初ISM的成像速度只有0.001 6 Hz@10 μm2。除此外，ISM需要借助數(shù)字式的光子重定位與減卷積算法實(shí)現(xiàn)超分辨圖像，重建算法的優(yōu)劣與成像過(guò)程中的任何因素都會(huì)影響重建圖像的質(zhì)量(見(jiàn)圖4a)。近幾年減卷積算法的優(yōu)化與硬件技術(shù)的革新使得PS-SIM技術(shù)在成像速度與成像質(zhì)量上都有了極大的提升[69-77]。將數(shù)字方式的光子重定位換成光學(xué)方式的光子重定位，便是OPRA/RE-scan，它們可在一次采集過(guò)程中實(shí)現(xiàn)超分辨成像，避免了多次采集原始圖像時(shí)間上的消耗，但仍然受點(diǎn)掃描技術(shù)的“像素停留時(shí)間”限制(見(jiàn)圖4b)。多點(diǎn)掃描提供了一種“并行激發(fā)”方式，可以避免單點(diǎn)掃描技術(shù)的“像素停留時(shí)間”限制，MSIM通過(guò)采用高速DMD產(chǎn)生的稀疏二維照明圖案來(lái)掃描樣品，雖然仍使用數(shù)字式光子重定位獲得超分辨圖像，卻使得成像速度提升到了1 Hz@48 μm2(見(jiàn)圖5a)，進(jìn)一步，將多點(diǎn)掃描與光學(xué)方式的光子重定位結(jié)合，iSIM將成像速度提升到了100 Hz，僅僅受相機(jī)讀出時(shí)間的限制(見(jiàn)圖5b)。

Tab.2 Implementation methods of PS-SIM technology

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.

共聚焦技術(shù)之所以一直是活細(xì)胞成像中的主力工具，除了比較合適的成像速度外就是相對(duì)高的分辨率。單光子激發(fā)的PS-SIM技術(shù)在結(jié)合多點(diǎn)掃描與光學(xué)方式的光子重定位后已經(jīng)完全超越共聚焦技術(shù)，不僅具有超分辨成像的能力，而且成像速度是現(xiàn)有共聚焦技術(shù)的幾倍以上，除此之外還保留了共聚焦技術(shù)的多色成像功能。借助多色超分辨成像，生物學(xué)家們可以更準(zhǔn)確的捕捉到活細(xì)胞中同一位置不同結(jié)構(gòu)之間的相互作用。多色成像主要有兩種實(shí)現(xiàn)方式，第一種是多譜線激發(fā)，對(duì)每個(gè)譜線激發(fā)的熒光信號(hào)進(jìn)行多次探測(cè)，將圖像融合實(shí)現(xiàn)多色成像，MSIM與iSIM便是采用這種方式。但是這種技術(shù)在多色成像過(guò)程中需要多次切換激光波長(zhǎng)，會(huì)不可避免地造成圖像上的空間錯(cuò)位，而且也不適合于鈣離子等的快速動(dòng)態(tài)成像。第二種是單色激發(fā)，多通道探測(cè)的方式。通過(guò)分光器件(例如光柵、棱鏡或者濾光片組)將不同波長(zhǎng)的熒光信號(hào)分離，雖然可以從不同的探測(cè)通道上收集分離的熒光信號(hào)，但由于熒光蛋白寬譜帶且相互交叉的激發(fā)與發(fā)射譜，這種技術(shù)不可避免地會(huì)造成融合圖像的光譜串?dāng)_。我們結(jié)合RE-scan技術(shù)與光譜解混(spectral unmixing)原理建立了一種光譜分辨的單光子激發(fā)超分辨顯微鏡，實(shí)現(xiàn)了單次激發(fā)條件下的多色超分辨顯微成像[78](見(jiàn)圖6)，不僅確保了多色融合后圖像的光譜純凈性，而且由于我們是一次成像便可獲得多色超分辨成像結(jié)果，該技術(shù)在鈣離子等的快速動(dòng)態(tài)成像中有很強(qiáng)的應(yīng)用價(jià)值。

Fig.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 圖6 基于光譜分辨的單光子激發(fā)超分辨顯微成像[78]。SYTO 82與LysoTracker Red分別標(biāo)記了bEnd3型活細(xì)胞的細(xì)胞核(圖中紅色)與溶酶體(圖中綠色)；(a,e)普通的RE-scan超分辨圖像； (d,h)基于光譜分辨的RE-scan超分辨圖像；(b,f)和(c,g)分別為光譜解混分離出的細(xì)胞核和溶酶體；(i)為兩種染料的熒光光譜

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技術(shù)被提出后最大的優(yōu)勢(shì)便是與雙光子激發(fā)相結(jié)合，將只能觀察細(xì)胞厚度的超分辨技術(shù)拓展為可以觀察組織厚度的超分辨技術(shù)[67-68,79]。無(wú)論是WF-SIM技術(shù)，基于單光子激發(fā)的PS-SIM技術(shù)，還是其他超分辨技術(shù)，一個(gè)最大的問(wèn)題就是激發(fā)光的光場(chǎng)分布與光強(qiáng)大小容易受組織散射的影響，隨著成像深度的增加，光場(chǎng)逐漸變形，光強(qiáng)逐漸變小。雙光子激發(fā)使用較長(zhǎng)波長(zhǎng)的近紅外激光作為激發(fā)光源，從根本上降低了組織對(duì)激發(fā)光的散射。基于雙光子激發(fā)的MSIM與OPRA/RE-scan在活體組織中實(shí)現(xiàn)了145 nm的橫向分辨率和400 nm的軸向分辨率，成像深度超過(guò)100 μm，成像速度接近1 Hz。目前，基于雙光子激發(fā)的iSIM并沒(méi)有文章報(bào)道，但理論上它也可以像單光子激發(fā)下的iSIM一樣快速，像基于雙光子激發(fā)的MSIM與OPRA/RE-scan一樣進(jìn)行大深度的組織超分辨成像。

Fig.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 圖7 結(jié)合自適應(yīng)光學(xué)的雙光子激發(fā)超分辨顯微成像[79]。a、b、c、d及e、f分別為普通雙光子激發(fā)超分辨顯微鏡、基于自適應(yīng)光學(xué)的雙光子激發(fā)超分辨顯微鏡與基于自適應(yīng)光學(xué)的雙光子激發(fā)超分辨顯微鏡，并結(jié)合圖像減卷積處理后的細(xì)胞骨架成像結(jié)果；g～l分別為e圖對(duì)應(yīng)區(qū)域的放大圖；m表示系統(tǒng)的橫向與縱向分辨率；n表示自適應(yīng)校正前后的波前相位圖

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.

雖然使用雙光子激發(fā)確實(shí)提高了PS-SIM技術(shù)在超分辨成像下的穿透深度，但是隨著成像深度的加深，激發(fā)光斑的形狀仍然會(huì)發(fā)生變形，相對(duì)的，在大深度成像時(shí)，激發(fā)出來(lái)的熒光信號(hào)也更容易受散射影響。為了解決這一問(wèn)題，我們?cè)赑S-SIM技術(shù)的基礎(chǔ)上引入了自適應(yīng)光學(xué)，提出了基于自適應(yīng)光學(xué)的雙光子激發(fā)超分辨顯微鏡[79](見(jiàn)圖7)。該系統(tǒng)同時(shí)具備超分辨光學(xué)顯微成像功能和大深度三維成像能力，使光學(xué)超分辨成像深度推進(jìn)至250 μm，橫向分辨率依然能保持在176 nm、縱向分辨率保持在729 nm。利用該技術(shù)，我們對(duì)細(xì)胞、線蟲(chóng)胚胎及幼蟲(chóng)、果蠅腦片和斑馬魚(yú)胚胎開(kāi)展了高清晰三維成像研究，成像效果遠(yuǎn)優(yōu)于傳統(tǒng)雙光子成像。由于該技術(shù)提高了光子利用效率，從而降低了所需激光功率，可以允許發(fā)育學(xué)家在長(zhǎng)達(dá)1個(gè)小時(shí)的連續(xù)三維成像中對(duì)線蟲(chóng)胚胎的發(fā)育過(guò)程開(kāi)展高清晰的三維動(dòng)態(tài)觀測(cè)。

4 Summary and outlook

總結(jié)與展望

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.

無(wú)論是在生命科學(xué)領(lǐng)域還是生物醫(yī)學(xué)領(lǐng)域，共聚焦顯微鏡一直是科研工作者的必備工具。近幾年，隨著超分辨技術(shù)在硬件與軟件上的完善，結(jié)構(gòu)光照明超分辨顯微鏡已成為目前最被看好的可以完全取代共聚焦顯微鏡的技術(shù)。活細(xì)胞成像層面，WF-SIM技術(shù)和單光子激發(fā)的PS-SIM技術(shù)可以提供遠(yuǎn)超共聚焦分辨率與成像速度的圖像信息?；铙w成像方面，結(jié)合雙光子激發(fā)的PS-SIM技術(shù)則可以提供大成像深度下的超分辨圖像信息，這是傳統(tǒng)共聚焦技術(shù)與雙光子技術(shù)都不具備的。除此之外，針對(duì)特殊的應(yīng)用，SIM技術(shù)還可以與其他成像技術(shù)融合，例如針對(duì)胚胎發(fā)育學(xué)，SIM技術(shù)與光片照明技術(shù)的融合，針對(duì)材料化學(xué)，SIM技術(shù)與等離子體結(jié)構(gòu)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.

經(jīng)過(guò)近20年的發(fā)展，WF-SIM技術(shù)在細(xì)胞成像應(yīng)用的潛力已經(jīng)被充分挖掘。一位有光學(xué)儀器開(kāi)發(fā)經(jīng)驗(yàn)的研究人員在經(jīng)歷短時(shí)間的系統(tǒng)構(gòu)建培訓(xùn)后花費(fèi)較少的財(cái)力與精力就可以搭建起一套2D-SIM或者3D-SIM系統(tǒng)。一位非光學(xué)專業(yè)的研究人員也可以在接受1～2天的樣品制備與系統(tǒng)標(biāo)定培訓(xùn)后使用WF-SIM系統(tǒng)獲得優(yōu)秀的超分辨圖像[57]。相比之下，提出至如今不到十年的PS-SIM技術(shù)卻還有很大的發(fā)展空間。(1)PS-SIM不僅可以很好的融合于現(xiàn)有共聚焦顯微鏡中，而且操作方便，但是需要進(jìn)一步提高分辨率。一種可能的實(shí)現(xiàn)方法是結(jié)合光開(kāi)關(guān)熒光蛋白，利用非線性熒光激發(fā)效應(yīng)，通過(guò)激活光第一次照射控制光開(kāi)關(guān)熒光蛋白被激活的可能性，再通過(guò)激發(fā)光激發(fā)這些待激發(fā)的蛋白就可以實(shí)現(xiàn)PS-SIM技術(shù)分辨率再提高。(2)PS-SIM技術(shù)可以完美的與雙光子技術(shù)結(jié)合，利用雙光子在生物組織的強(qiáng)抗散射能力實(shí)現(xiàn)250 μm成像深度下的雙光子超分辨成像。但這與普通雙光子技術(shù)報(bào)道的1 mm成像深度相比還是極為有限。PS-SIM技術(shù)可進(jìn)一步結(jié)合三光子技術(shù)實(shí)現(xiàn)更大探測(cè)深度的三光子超分辨成像。除此之外，PS-SIM技術(shù)還可以與光聲，OCT等新型大深度成像技術(shù)結(jié)合，充分發(fā)揮各技術(shù)的優(yōu)勢(shì)，實(shí)現(xiàn)各技術(shù)分辨率與成像深度的新突破。(3)進(jìn)一步推進(jìn)基于雙光子激發(fā)的iSIM實(shí)現(xiàn)，在提升分辨率與探測(cè)深度的前提下，進(jìn)一步提升顯微鏡的三維成像(體成像)速度，使這一技術(shù)在神經(jīng)科學(xué)，免疫學(xué)等的研究中發(fā)揮前所未有的效用。

參考文獻(xiàn)：

[1] ABBE E. Contributions to the theory of the microscope and that microscopic perception[J].Arch.Microsc.Anat,1873,9:413-468.

[2] HUANG B,BATES M,ZHUANG X. Super-resolution fluorescence microscopy[J].AnnualReviewofBiochemistry,2009,78:993-1016.

[3] SCHERMELLEH L,HEINTZMANN R,LEONHARDT H. A guide to super-resolution fluorescence microscopy[J].TheJournalofCellBiology,2010,190(2):165-175.

[4] HELL S W,WICHMANN J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy[J].OpticsLetters,1994,19(11):780-782.

[5] KLAR T A,JAKOBS S,DYBA M,etal.. Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission[J].ProceedingsoftheNationalAcademyofSciences,2000,97(15):8206-8210.

[6] BETZIG E,PATTERSON G H,SOUGRAT R,etal.. Imaging intracellular fluorescent proteins at nanometer resolution[J].Science,2006,313(5793):1642-1645.

[7] HESS S T,GIRIRAJAN T P K,MASON M D. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy[J].BiophysicalJournal,2006,91(11):4258-4272.

[8] SHROFF H,GALBRAITH C G,GALBRAITH J A,etal.. Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics[J].NatureMethods,2008,5(5):417-423.

[9] RUST M J,BATES M,ZHUANG X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy(STORM)[J].NatureMethods,2006,3(10):793-795.

[10] BATES M,HUANG B,DEMPSEY G T,etal.. Multicolor super-resolution imaging with photo-switchable fluorescent probes[J].Science,2007,317(5845):1749-1753.

[11] HUANG B,WANG W,BATES M,etal.. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy[J].Science,2008,319(5864):810-813.

[12] HEINTZMANN R,CREMER C. Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating[J].Proc.SPIE,1999,3568:185-196.

[13] GUSTAFSSON M G L,AGARD D A,SEDAT J W. Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination[J].Proc.SPIE,2000,3919:141-150.

[14] GUSTAFSSON M G L. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy[J].JournalofMicroscopy,2000,198(2):82-87.

[15] WICKER K. Super-resolution fluorescence microscopy using structured illumination[J].Super-ResolutionMicroscopyTechniquesintheNeurosciences,2014:133-165.

[16] REGO E H,SHAO L. Practical structured illumination microscopy[J].AdvancedFluorescenceMicroscopy:MethodsandProtocols,2015:175-192.

[17] FROHN J T,KNAPP H F,STEMMER A. True optical resolution beyond the Rayleigh limit achieved by standing wave illumination[J].ProceedingsoftheNationalAcademyofSciences,2000,97(13):7232-7236.

[18] GUSTAFSSON M G L,SHAO L,CARLTON P M,etal.. Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination[J].BiophysicalJournal,2008,94(12):4957-4970.

[19] HEINTZMANN R,JOVIN T M,CREMER C. Saturated patterned excitation microscopy-a concept for optical resolution improvement[J].JOSAA,2002,19(8):1599-1609.

[20] GUSTAFSSON M G L. Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution[J].ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica,2005,102(37):13081-13086.

[21] HIRVONEN L,MANDULA O,WICKER K,etal.. Structured illumination microscopy using photoswitchable fluorescent proteins[J].Proc.SPIE,2008,6861:68610L.

[22] REGO E H,SHAO L,MACKLIN J J,etal.. Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution[J].ProceedingsoftheNationalAcademyofSciences,2012,109(3):E135-E143.

[23] SCHERMELLEH L,CARLTON P M,HAASE S,etal.. Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy[J].Science,2008,320(5881):1332-1336.

[24] KNER P,CHHUN B B,GRIFFIS E R,etal.. Super-resolution video microscopy of live cells by structured illumination[J].NatureMethods,2009,6(5):339-342.

[25] SHAO L,KNER P,REGO E H,etal.. Super-resolution 3D microscopy of live whole cells using structured illumination[J].Naturemethods,2011,8(12):1044.

[26] LI D,SHAO L,CHEN B C,etal.. Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics[J].Science,2015,349(6251):aab3500.

[27] CHANG B J,CHOU L J,CHANG Y C,etal.. Isotropic image in structured illumination microscopy patterned with a spatial light modulator[J].OpticsExpress,2009,17(17):14710-14721.

[28] DAN D,LEI M,YAO B,etal.. DMD-based LED-illumination Super-resolution and optical sectioning microscopy[J].ScientificReports,2013,3.

[30] F?RSTER R,LU-WALTHER H W,JOST A,etal.. Simple structured illumination microscope setup with high acquisition speed by using a spatial light modulator[J].OpticsExpress,2014,22(17):20663-20677.

[31] LU-WALTHER H W,KIELHORN M,F RSTER R,etal.. fastSIM:a practical implementation of fast structured illumination microscopy[J].MethodsandApplicationsinFluorescence,2015,3(1):014001.

[32] SONG L,LU-WALTHER H W,F RSTER R,etal.. Fast structured illumination microscopy using rolling shutter cameras[J].MeasurementScienceandTechnology,2016,27(5):055401.

[33] YOUNG L J,STR?HL F,KAMINSKI C F. A guide to structured illumination TIRF microscopy at high speed with multiple colors[J].JournalofVisualizedExperiments:JoVE,2016(111).

[34] 文剛,李思黽,楊西斌,等.基于激光干涉的結(jié)構(gòu)光照明超分辨熒光顯微鏡系統(tǒng)[J].光學(xué)學(xué)報(bào),2017,37(3):32-42.

WEN G,LI S M,YANG X B,etal.. Super-resolution fluorescence microscopy system by structured illumination based on laser interference[J].ChineseJournalofOptics,2017,37(3):32-42.(in Chinese)

[35] SCHAEFER L H,SCHUSTER D,SCHAFFER J. Structured illumination microscopy: artefact analysis and reduction utilizing a parameter optimization approach[J].JournalofMicroscopy,2004,216(2):165-174.

[36] SHROFF S A,FIENUP J R,WILLIAMS D R. Phase-shift estimation in sinusoidally illuminated images for lateral superresolution[J].JOSAA,2009,26(2):413-424.

[37] SHROFF S A,FIENUP J R,WILLIAMS D R. Lateral superresolution using a posteriori phase shift estimation for a moving object: experimental results[J].JOSAA,2010,27(8):1770-1782.

[38] O'HOLLERAN K,SHAW M. Polarization effects on contrast in structured illumination microscopy[J].OpticsLetters,2012,37(22):4603-4605.

[39] WICKER K. Non-iterative determination of pattern phase in structured illumination microscopy using auto-correlations in Fourier space[J].OpticsExpress,2013,21(21):24692-24701.

[40] WICKER K,MANDULA O,BEST G,etal.. Phase optimisation for structured illumination microscopy[J].OpticsExpress,2013,21(2):2032-2049.

[41] WICKER K. Non-iterative determination of pattern phase in structured illumination microscopy using auto-correlations in Fourier space[J].OpticsExpress,2013,21(21):24692-24701.

[42] AYUK R,GIOVANNINI H,JOST A,etal.. Structured illumination fluorescence microscopy with distorted excitations using a filtered blind-SIM algorithm[J].OpticsLetters,2013,38(22):4723-4726.

[43] O'HOLLERAN K,SHAW M. Optimized approaches for optical sectioning and resolution enhancement in 2D structured illumination microscopy[J].BiomedicalOpticsExpress,2014,5(8):2580-2590.

[44] CHU K,MCMILLAN P J,SMITH Z J,etal.. Image reconstruction for structured-illumination microscopy with low signal level[J].OpticsExpress,2014,22(7):8687-8702.

[45] CHAKROVA N,HEINTZMANN R,RIEGER B,etal.. Studying different illumination patterns for resolution improvement in fluorescence microscopy[J].OpticsExpress,2015,23(24):31367-31383.

[47] BALL G,DEMMERLE J,KAUFMANN R,etal.. SIMcheck: a toolbox for successful super-resolution structured illumination microscopy[J].ScientificReports,2015,5.

[48] F?RSTER R,WICKER K,MüLLER W,etal.. Motion artefact detection in structured illumination microscopy for live cell imaging[J].OpticsExpress,2016,24(19):22121-22134.

[49] ZHOU X,LEI M,DAN D,etal.. Image recombination transform algorithm for superresolution structured illumination microscopy[J].JournalofBiomedicalOptics,2016,21(9):096009-096009.

[50] CHAKROVA N,RIEGER B,STALLINGA S. Deconvolution methods for structured illumination microscopy[J].JOSAA,2016,33(7):B12-B20.

[51] PEREZ V,CHANG B J,STELZER E H K. Optimal 2D-SIM reconstruction by two filtering steps with Richardson-Lucy deconvolution[J].ScientificReports,2016,6:37149.

[52] YANG Q,CAO L,ZHANG H,etal.. Method of lateral image reconstruction in structured illumination microscopy with super resolution[J].JournalofInnovativeOpticalHealthSciences,2016,9(3):1630002.

[53] LAL A,SHAN C,XI P. Structured illumination microscopy image reconstruction algorithm[J].IEEEJournalofSelectedTopicsinQuantumElectronics,2016,22(4):50-63.

[54] MüLLER M,M?NKEM?LLER V,HENNIG S,etal.. Open-source image reconstruction of super-resolution structured illumination microscopy data in ImageJ[J].NatureCommunications,2016,7.

[55] 周興,但旦,千佳,等.結(jié)構(gòu)光照明顯微中的超分辨圖像重建研究[J].光學(xué)學(xué)報(bào),2017,37(3):10-21.

ZHOU X,DAN D,QIAN J,etal.. Super-resolution reconstruction theory in structured illumination microscopy[J].ChineseJournalofOptics,2017,37(3):10-21.(in Chinese)

[56] 趙天宇,周興,但旦,等.結(jié)構(gòu)光照明顯微中的偏振控制[J].物理學(xué)報(bào),2017,66(14):295-305.

ZHAO T Y,ZHOU X,DAN D,etal.. Polarization control methods in structured illumination microscopy[J].ChineseJournalofPhysics,2017,66(14):295-305.(in Chinese)

[57] DEMMERLE J,INNOCENT C,NORTH A J,etal.. Strategic and practical guidelines for successful structured illumination microscopy[J].Nat.Protoc,2017.

[58] KRAUS F,MIRON E,DEMMERLE J,etal.. Quantitative 3D structured illumination microscopy of nuclear structures[J].NatureProtocols,2017,12(5):1011-1028.

[59] STR?HL F,KAMINSKI C F. Frontiers in structured illumination microscopy[J].Optica,2016,3(6):667-677.

[60] MüLLER C B,ENDERLEIN J. Image scanning microscopy[J].PhysicalReviewLetters,2010,104(19):198101.

[61] YORK A G,PAREKH S H,DALLENOGARE D,etal.. Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy[J].NatureMethods,2012,9(7):749-754.

[62] SCHULZ O,PIEPER C,CLEVER M,etal.. Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy[J].ProceedingsoftheNationalAcademyofSciences,2013,110(52):21000-21005.

[63] SHEPPARD C J R,MEHTA S B,HEINTZMANN R. Superresolution by image scanning microscopy using pixel reassignment[J].OpticsLetters,2013,38(15):2889-2892.

[64] DE LUCA G M R,BREEDIJK R M P,BRANDT R A J,etal.. Re-scan confocal microscopy: scanning twice for better resolution[J].BiomedicalOpticsExpress,2013,4(11):2644-2656.

[65] ROTH S,SHEPPARD C J R,WICKER K,etal.. Optical photon reassignment microscopy(OPRA)[J].OpticalNanoscopy,2013,2(1):5.

[66] YORK A G,CHANDRIS P,DALLENOGARE D,etal.. Instant super-resolution imaging in live cells and embryos via analog image processing[J].NatureMethods,2013,10(11):1122-1126.

[67] INGARAMO M,YORK A G,WAWRZUSIN P,etal.. Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue[J].ProceedingsoftheNationalAcademyofSciences,2014,111(14):5254-5259.

[68] WINTER P W,YORK A G,DALLENOGARE D,etal.. Two-photon instant structured illumination microscopy improves the depth penetration of super-resolution imaging in thick scattering samples[J].Optica,2014,1(3):181-191.

[69] WEISSHART K. The basic principle of airyscanning[J].ZeissTechnologyNote,2014:22.

[70] STR?HL F,KAMINSKI C F. A joint Richardson-Lucy deconvolution algorithm for the reconstruction of multifocal structured illumination microscopy data[J].MethodsandApplicationsinFluorescence,2015,3(1):014002.

[71] AZUMA T,KEI T. Super-resolution spinning-disk confocal microscopy using optical photon reassignment[J].OpticsExpress,2015,23(11):15003-15011.

[72] CURD A,CLEASBY A,MAKOWSKA K,etal.. Construction of an instant structured illumination microscope[J].Methods,2015,88:37-47.

[73] MCGREGOR J E,MITCHELL C A,HARTELL N A. Post-processing strategies in image scanning microscopy[J].Methods,2015,88:28-36.

[74] SIVAGURU M,URBAN M A,FRIED G,etal.. Comparative performance of airyscan and structured illumination superresolution microscopy in the study of the surface texture and 3D shape of pollen[J].MicroscopyResearchandTechnique,2016.

[75] ROTH S,HEINTZMANN R. Optical photon reassignment with increased axial resolution by structured illumination[J].MethodsandApplicationsinFluorescence,2016,4(4):045005.

[76] SHEPPARD C J R,ROTH S,HEINTZMANN R,etal.. Interpretation of the optical transfer function: Significance for image scanning microscopy[J].OpticsExpress,2016,24(24):27280-27287.

[77] DE LUCA G M R,DESCLOS E,BREEDIJK R M P,etal.. Configurations of the Re-scan Confocal Microscope(RCM) for biomedical applications[J].JournalofMicroscopy,2017,266(2):166-177.

[78] CHEN L C,ZHENG W,etal.. Multicolor re-scan super-resolution imaging of live cells(unpublished).

[79] ZHENG W,WU Y,WINTER P,etal.. Adaptive optics improves multiphoton super-resolution imaging[J].NatureMethods,2017,14(9):869-872.