Super-resolution Optical Microscopic Imaging Based On STED

Due to its diffraction limit restrictions, conventional optical microscopes can only achieve spatial resolution of 200 nm, thus biologists can't look into cells by this technology to explore many cellular compartments and proteins whose sizes are smaller than 200 nm.At present, some super-resolution microscopy technologies, such as Photoactivated Localization Microscopy(PALM), Stochastic Optical Reconstruction microscopy(STORM) and Stimulated Emission Depletion(STED), have been developed in order to break diffraction limit restrictions. The STED imaging technology takes advantages of breaking the light limitation, flexible choices of fluorescence probe and real-time imaging in a living cell. Therefore, it has broad prospect to being applied in the research of biology field. In this paper, we mainly study the imaging method of the super-resolution optical microscopy. The main contents are as following:We simulate the imaging of STED, derivate the intensity formulas of the depletion light beams when an incident light are the linearly polarized light and a radially polarized light. In order to visually observe the influence of numerical aperture(NA) to the resolution of STED systems, we simulate the full width half maximum(FWHM) with different incident lights and different number of NA. We first adopt the linearly polarized light as the incident light. The simulation result shows the FWHM becomes narrower changing from 0.82 to 0.52 when NA increasing from 0.7 to 1.1, however, when NA continues to increase from 1.1 to 1.3, the FWHM becomes increased conversely.When the incident light is radially polarized light, the simulation result shows the FWHM becomes narrower changing from 0.5 to 0.26 when NA increasing from 0.7 to 1.1. Comparing with the case of the incident light being linearly polarized light, we can get smaller FWHM, or the doughnut-shaped spot becoming smaller when the incident light is radially polarized light. These results show that the FWHM obtained by using an radial polarization light as an input beam is smaller than that obtained by using the linearly polarized light under the same NA. So, applying the radially polarized light as the illumination in STED microscopy can get the doughnut-shaped beam which is radially distributed. The doughnut-shaped beam can improve the imaging-resolution of STED microscopy.We design the depletion optical path in which the light beam passing through the vortex phase plate(VPP) is focused by the high numerical aperture after phase coding, finally generating doughnut-shaped beam in the focal plane. In order to verify the feasibility of the depletion optical path, we simulate the experimental light path by the Zemax software, the simulation results show that we can form a doughnut-shaped beam in the depletion optical path. Finally, we set up the depletion optical path and conduct a preliminary experiment to generate a doughnut-shaped beam. Through the further experiment, we verify the doughnut-shaped beam is radially distributed.Finally, we simulate the intensity distribution when we add the depletion light to the excitation light, furthermore we analysis the influence of numerical aperture(NA) to the intensity distribution.