Abstract
We report three-dimensional fluorescence emission difference (3D-FED) microscopy using a spatial light modulator (SLM). Zero phase, 0–2[Formula: see text] 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 differently 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[Formula: see text]nm fluorescent beads. The experiment yields lateral resolution of 140[Formula: see text]nm and axial resolution of approximate 380[Formula: see text]nm.
Highlights
Far- ̄eld °uorescence microscopy has been applied in biological and medical science to observe the microstructures and their movements[1] owing to its simplicity, versatility, and noninvasiveness
We present a simple approach to achieve 3D super-resolution imaging using Fluorescence emission di®erence (FED), and we call it the 3D-FED
In 3D-FED, three di®erent confocal scanning images are required to obtain thenal 3D-FED image: the confocal image is acquired under the solid excitation pattern, the lateral negative confocal image is acquired under the doughnut excitation pattern,[7] and the axial-negative confocal image is acquired under the z-axis hollow excitation pattern
Summary
Far- ̄eld °uorescence microscopy has been applied in biological and medical science to observe the microstructures and their movements[1] owing to its simplicity, versatility, and noninvasiveness. Fluorescence emission di®erence (FED)[6,7] microscopy was recently reported as a novel superresolution technique It is based on intensity subtraction[8] between two images acquired under solid and hollow illumination patterns. Our technique achieves super-resolved image by subtracting three di®erently acquired images with the proper factors according to the simulation and experiment situation.[19,24,25,26,27] Images of °uorescent beads on a coverslip prove that this technique can be implemented successfully in confocal microscopy and improves the lateral and the axial resolution
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