几种超分辨率荧光显微技术的原理和近期进展

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200 nm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(point spread function, 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(photoactivated localization microscopy, PALM)和随机光学重构显微技术(stochastic optical reconstruction microscopy, 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(stimulated emission depletion, STED)和饱和结构照明显微技术(saturated structure illumination microscopy, 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．

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

In life science research, it is often required to localize proteins in a live cell to a certain accuracy to study their localization-related function. However, due to the Abbe/Rayleigh criteria of light, the widely used wide-field/confocal microscopy can never resolve structures less than 200 nm in diameter. In recent year, different super-resolution microscopy techniques emerge as a result of new fluorescent probes and imaging theories. A full frame to the theories and recent advancements in this field is summarized. The concept of point-spread function of light source in the focal plane and the classical definition of resolution is explained in the first part. The fluorescence single-molecular imaging technique and the equation that defines the localization accuracy of a single molecule is introduced in the second part. Based on these knowledge, super-resolution microscopy methods based on single-molecular imaging technique, such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) is discussed further. On the other hand, by engineering the point spread function of the light source, super-resolution can also be achieved. Two typical methods, stimulated emission depletion (STED) and saturated structure illumination microscopy (SSIM) are explored thereafter. In the end, different methods to extract super-resolutional information along the z axis, and their combinations with the methods to increase xy plane resolution mentioned above are explained. In the end, the limitation to the current super-resolution methods and their future direction are also discussed.