Rethinking resolution estimation in fluorescence microscopy: from theoretical resolution criteria to super-resolution microscopy

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.