Introduction to Theories of Several Super-resolution Fluorescence Microscopy Methods and Recent Advance in The Field*

Abstract

几种超分辨率荧光显微技术的原理和近期进展 * 吕志坚 1) 陆敬泽 2) 吴雅琼 1) 陈良怡 2)** ( 1) 北京市科学技术情报研究所，北京 100044； 2) 中国科学院生物物理研究所，北京 100101) 摘要 在生命科学领域，人们常常需要在细胞内精确定位特定的蛋白质以研究其位置与功能的关系． 多年来，宽场 / 共聚焦 荧光显微镜的分辨率受限于光的阿贝 / 瑞利极限，不能分辨出 200nm 以下的结构． 近年来，随着新的荧光探针和成像理论 的出现，研究者开发了多种实现超出普通共聚焦显微镜分辨率的三维超分辨率成像方法． 主要介绍这些方法的原理、近期进 展和发展趋势． 介绍了光源的点扩散函数(pointspreadfunction,PSF)的概念和传统分辨率的定义，阐述了提高 xy 平面分辨率 的方法． 通过介绍单分子荧光成像技术，引入了单分子成像定位精度的概念，介绍了基于单分子成像的超分辨率显微成像方 法 ， 包括光激活定位显微技术(photoactivatedlocalizationmicroscopy,PALM) 和随机光学重构显微技术(stochasticoptical reconstructionmicroscopy,STORM)． 介绍了两大类通过改造光源的点扩散函数来提高成像分辨率的方法，分别是受激发射损 耗显微技术(stimulatedemissiondepletion,STED)和饱和结构照明显微技术(saturatedstructureilluminationmicroscopy,SSIM)． 比较了不同的 z 轴提取信息的方法，并阐述了这些方法与 xy 平面上的超分辨率显微成像技术相结合所得到的各种三维超分 辨率显微成像技术的优劣． 探讨了目前超分辨率显微成像的发展极限和方向． 关键词 超分辨率荧光显微技术，点扩散函数，PALM，STORM，STED，SSIM 学科分类号 Q27，Q63

Nucleic Acid Probes for Single-Molecule Localization Imaging of Cellular Biomolecules

“…28,29 SMLM can overcome the diffraction-limited resolution barrier of optical microscopy, achieving super resolution imaging. 30 As shown in Figure 1, the core ideas of SMLM are "blinking", "localization," and "reconstruction". 5 The "blinking" is usually based on specific fluorescent probes.…”

“…STORM has been developed to solve this problem. 30 As illustrated in Figure 2B, Cy3−Cy5 dye pairs are used to label endogenous proteins in this technique. 43 The photoswitch of Cy5 can be realized by switching between the 633 nm wavelength laser and the 532 nm wavelength laser.…”

“…Although the super-resolution visualization of cellular foreign proteins can be achieved using PALM, the imaging of intracellular proteins cannot be done. STORM has been developed to solve this problem . As illustrated in Figure B, Cy3–Cy5 dye pairs are used to label endogenous proteins in this technique .…”