陈廷爱，陈龙超，李 慧，余 佳，高玉峰，郑 炜*

(1.中国科学院 深圳先进技术研究院，生物医学光学与分子影像研究室，广东 深圳 518055;2.睿芯生命科技(深圳)有限公司，广东 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)；……