Generation and comparison of donut-shaped depletion beams in STED microscopy

Abstract

The profile of donut-shaped depletion beam focused by a high numerical aperture (NA) microscope objective determines the resolution and signal to noise ratio (SNR) of image in stimulation emission depletion (STED) microscopy. Thus, the effects of different donut-shaped depletion beams on the STED microscopy are needed to be researched. In this paper, theoretical and experimental researches have been conducted on the effect of donut-shaped depletion beams on the resolution and SNR of image in stimulation emission depletion (STED) microscopy. Four kinds of donut-shaped depletion beams had been researched, including linear polarization with vortex phase, left circular polarization with vortex phase, right circular polarization with vortex phase and azimuthal polarization. Simulations of effective fluorescence point spread function (PSF) had been conducted with the different donut-shaped depletion beams mentioned above. The peak to peak distance for azimuthal polarization minimizes about 10% than right circular polarization. The donut-shaped beams were experimental tested. The effective fluorescence PSF was measured by fluorescence beads, evidencing a spatial resolution of 36nm in the focal plane. The results show that depletion beams of the right circular polarization combined with vortex phase and the azimuthal polarization are preferable for STED microscopy. Furthermore, higher resolution could be achieved with the azimuthally polarized depletion beam, while the right circularly polarized depletion beam could be generated more easily in the experiments.