Enhancement of fluorescence emission difference microscopy using conjugated vortex phase modulation

In this paper, we propose and demonstrate an improved fluorescence emission difference microscopy (FED) method, exploiting programmable phase modulation for image enhancement. The main novelty of the proposed approach lies in the matched size and intensity of two excitation spots. The proposed method improves the FED performance on image quality via artefact elimination. We demonstrate the feasibility of this method through theoretical studies and experimental tests. The experimental results of nanobeads and cells validate the practical performance of this method, which can enable reliable observations at superresolution in biomedical studies. LAY DESCRIPTION: In this paper, we propose a method to improve the imaging quality of regular fluorescence emission difference (FED) microscopy. In regular FED imaging, a solid and a doughnut excitation beam are successively used to acquire two images which are then subtracted with each other to improve the resolution of confocal microscopy. The doughnut beam can be generated by modulating the excitation beam with a vortex phase mask. Note that both of the excitation beam and the vortex phase mask must have the same handed direction in regular FED microscopy. However, some negative values may be produced and some information may be lost due to the subtraction process in regular FED imaging, which is mainly caused by the mismatched size and intensity of these two excitation spots. To address this issue, we propose conjugated FED (cFED) microscopy which additionally uses a conjugated vortex phase mask to modulate the solid beam to extend its focal spot size to be matched with the doughnut spot, which means the handed direction of the solid beam and the vortex phase mask is different. Besides, in order not to damage the resolution, the doughnut beam needs to be saturated to some degree. The experiment results show that, at the same resolution level, the negative values and the information loss in cFED image are all less than that of regular FED image.