Stimulated emission depletion microscopy with an offset depleting beam

We demonstrate that an offset stimulated emission depletion (STED) beam breaks the diffraction barrier of fluorescence microscopy in both the lateral and the axial directions. A 2.5-fold axial reduction of the focal spot is accomplished through the ear-shaped lobes of the diffraction maximum of the STED beam. The effect of the minima and side maxima of the STED beam on the lateral and axial resolution is shown to be in remarkable agreement with theory. Conditions are given for which a regular STED beam reduces the axial extent of a confocal spot from 490 +/- 36 to 175 +/- 18 nm, and simultaneously from 183 +/- 12 to 70 +/-8 nm along the direction of the offset. The latter establishes the lowest reported value in far-field fluorescence microscopy. (C) 2001 American Institute of Physics.