We propose a method for eliminating the deformations in fluorescence emission difference microscopy (FED). Due to excessive subtraction, negative values are inevitable in the original FED method, giving rise to deformations. We propose modulating the beam to generate an extended solid focal spot and a hollow focal spot. Negative image values can be avoided by using these two types of excitation spots in FED imaging. Hence, deformations are eliminated, and the signal-to-noise ratio is improved. In deformation-free imaging, the resolution is higher than that of confocal imaging by 32%. Compared to standard FED imaging with the same level of deformations, our method provides superior resolution.

What we believe is a novel method for improving confocal microscopy's resolution and contrast in 3D space is proposed. Based on a conventional confocal microscopy setup, we use an array detector composed of 32 photomultiplier tubes (PMTs) to replace one point-detector, where the location offset of each PMT caused a different effective point spread function (PSF). By applying array detection and the fluorescence emission difference method of an image with a solid PSF and another with a donut-shaped PSF, we can enhance lateral resolution about 27% in real time with only one scan, and improve the axial resolving ability by about 22% simultaneously. Experimental results of both fluorescent beads and living cells are presented to verify the applicability and effectiveness of our method.

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We report three-dimensional°uorescence emission di®erence (3D-FED) microscopy using a spatial light modulator (SLM). Zero phase, 0-2 vortex phase and binary 0-pi phase are loaded on the SLM to generate the corresponding solid, doughnut and z-axis hollow excitation spot, respectively. Our technique achieves super-resolved image by subtracting three di®erently acquired images with proper subtractive factors. Detailed theoretical analysis and simulation tests are proceeded to testify the performance of 3D-FED. Also, the improvement of lateral and axial resolution is demonstrated by imaging 100 nm°uorescent beads. The experiment yields lateral resolution of 140 nm and axial resolution of approximate 380 nm.

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