How low can STED go? Comparison of different write-erase beam combinations for stimulated emission depletion microscopy.

We compare different beam combinations for stimulated emission depletion microscopy. The four considered copolarized, mutually symmetric, but complementary write + erase beam combinations are circularly polarized beam + circularly polarized vortex with charge +1 or -1, azimuthally polarized with a vortex + azimuthally polarized, and radially polarized beam + radially polarized with a vortex. The resulting fluorescent spot was calculated for plane incident pump and erase beams, for plane waves with added high NA annular ring apertures, and when both incident beams were optimized with amplitude-phase masks. For all three incident wave cases, the azimuthal polarization combination consistently produces spots 15%-30% smaller than the commonly used, circularly polarized light combination (the first from above). The two other polarization combinations produce even smaller, of the order of nanometers/0.003λ, fluorescent spots with a caveat of having nonzero erase beam intensity in the center. Nevertheless, these combinations can be advantageous when exploiting PF, i.e., using molecules that respond solely to the longitudinal (or only to transversal) component of the illuminating field.