Three-dimensional resolution enhancement in confocal microscopy with radially polarized illumination using subtractive imaging

A method based on the subtraction of images taken under radially polarized illumination is provided to improve the three-dimensional resolution in confocal microscopy. Radially polarized light and vortex phase modulated radially polarized light are used as confocal microscopy illumination light. Our numerical simulations predict that lateral resolutions close to 0.218A is possible for practical confocal laser scanning microscopy with visible light. Theoretically, by applying radially polarized light and image subtraction method, we can enhance lateral resolution 47.3% higher than that in confocal microscopy with a pinhole of 0.3AU, and improve the axial resolving ability by 20.9% simultaneously. Simulation results of both 3D array model and cell microtubules are presented to verify the applicability and effectiveness of our method. In conclusion, the resolution of confocal microscopy using radially polarized light illumination and image subtraction could be improved. Compared with other resolution enhancement technologies, this method can offer significant spatial resolution improvements for fluorescence imaging at lower cost.