Phase filter enhanced STED-4Pi fluorescence microscopy: theory and experiment

Abstract

We study the point-spread and optical transfer-function (OTF) of a stimulated emission depletion (STED)-4Pi fluorescence microscope that provides diffraction unlimited resolution along the optic axis. Our calculations take into account the orientation of the linear transition dipole moment of the fluorescent molecules with respect to that of the focal field. We demonstrate a subdiffraction axial resolution of 44–48 nm for water-immersion lenses, corresponding to a 7–8-fold expansion of the OTF beyond the diffraction barrier of a single lens confocal microscope, which is in excellent agreement with theoretical predictions for the conditions applied. Furthermore, we study phase modifications of the wavefront of the stimulating beam that strengthen weakly transferred frequencies within the OTF support. The enlarged bandwidth enables the separation of objects at 76 nm axial distance.