Improvement of Imaging Resolution of Fluorescence Emission Difference Super-Resolution Microscopy with Annular Pupil

Super-resolution imaging can be achieved by fluorescence emission difference (FED) microscopy through the subtraction of a negative confocal image scanned by a hollow focal spot from a confocal image scanned by a solid focal spot. This study proposes an easily accessible annular pupil filter to enhance the resolution of deformation-free FED imaging. The hollow spot size is reduced by an appropriate annular pupil filter in the light path of hollow spot. Meanwhile, a diaphragm with a high numerical aperture is permitted for the beam of a solid focal spot, and a small solid focal spot which is matching well with the hollow focal spot is achieved. The spatial resolution of FED microscopy is increased under the effects of these two aspects.