We report three-dimensional fluorescence emission difference(3D-FED)microscopy using a spatial light modulator(SLM).Zero phase,0–2vortex 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.
Guangyuan ZhaoZihao RongCuifang KuangCheng ZhengXu Liu
Fourier ptychographic microscopy(FPM)is a newly developed imaging technique which stands out by virtue of its high-resolution and wide FOV.It improves a microscope's imaging perfor-mance beyond the diffraction limit of the employed optical components by illuminating the sample with oblique waves of different incident angles,similar to the concept of synthetic aperture.We propose to use an objective lens with high-NA to generate oblique illuminating waves in FPM.We demonstrate utilizing an objective lens with higher NA to iluminate the sample leads to better resolution by simulations,in which a resolution of 0.28 pum is achieved by using a high-NA illuminating objective lens(NA=1.49)and a low-NA collecting objective lens(NA=0.2)in coherent imaging(λ=488 nm).We then deeply study FPM's exact relevance of convergence speed to spatial spectrum overlap in frequency domain.The simulation results show that an overlap of about 60%is the optimal choice to acquire a high-quality recovery(520*520 pixels)with about 2 min's computing time.In addition,we testify the robustness of the algorithm of FPM to additive noises and its suitability for phase objects,which further proves FPM's potential application in biomedical imaging.
