Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan, 430074, China.
MoE Key Laboratory for Biomedical Photonics, Collaborative Innovation Center for Biomedical Engineering, School of Engineering Sciences, Huazhong University of Science and Technology, Wuhan, 430074, China.
Sci China Life Sci. 2020 Dec;63(12):1776-1785. doi: 10.1007/s11427-020-1785-4. Epub 2020 Dec 1.
Resolution is undoubtedly the most important parameter in optical microscopy by providing an estimation on the maximum resolving power of a certain optical microscope. For centuries, the resolution of an optical microscope is generally considered to be limited only by the numerical aperture of the optical system and the wavelength of light. However, since the invention and popularity of various advanced fluorescence microscopy techniques, especially super-resolution fluorescence microscopy, many new methods have been proposed for estimating the resolution, leading to confusions for researchers who need to quantify the resolution of their fluorescence microscopes. In this paper, we firstly summarize the early concepts and criteria for predicting the resolution limit of an ideal optical system. Then, we discuss some important influence factors that deteriorate the resolution of a certain fluorescence microscope. Finally, we provide methods and examples on how to measure the resolution of a fluorescence microscope from captured fluorescence images. This paper aims to answer as best as possible the theoretical and practical issues regarding the resolution estimation in fluorescence microscopy.
分辨率无疑是光学显微镜中最重要的参数，它可以估算出某种光学显微镜的最大分辨率。几个世纪以来，光学显微镜的分辨率通常被认为仅受限于光学系统的数值孔径和光的波长。然而，自从各种先进的荧光显微镜技术，特别是超分辨率荧光显微镜的发明和普及以来，已经提出了许多用于估计分辨率的新方法，这导致需要量化其荧光显微镜分辨率的研究人员感到困惑。在本文中，我们首先总结了预测理想光学系统分辨率极限的早期概念和标准。然后，我们讨论了一些会降低特定荧光显微镜分辨率的重要影响因素。最后，我们提供了从捕获的荧光图像中测量荧光显微镜分辨率的方法和示例。本文旨在尽可能地回答有关荧光显微镜分辨率估计的理论和实际问题。
