Precise background subtraction in stimulated emission double depletion nanoscopy.

Low-resolution background in stimulated emission depletion (STED) nanoscopy can arise from incomplete depletion or re-excitation by the STED beam. We have recently introduced stimulated emission double depletion (STEDD), a technique to efficiently suppress this background. In STEDD, the conventional, doughnut-shaped STED pulse, which depletes excited fluorophores outside the center of the focal region, is followed by a second Gaussian STED pulse, which specifically depletes the central region. The background is removed by calculating a weighted difference of photon events collected before and after the second STED pulse. Here, we present a simple, yet powerful, method to determine the weight factor, which depends on the fluorescence decay, from a direct analysis of the acquired data. We vary the weight factor to identify its optimal value as the one for which the weight of high-frequency components in the spectrum of the acquired STEDD image is maximized. This strategy is also applicable to other differential approaches for background suppression in imaging.