Point-spread function optimization in isoSTED nanoscopy.

IsoSTED nanoscopy, a variant of stimulated emission depletion (STED) microscopy, utilizes two opposing objective lenses and features the highest three-dimensional resolution of STED nanoscopes currently available. However, this technique is limited by axially repetitive side minima in the interference pattern of the depletion point-spread function (PSF), which can lead to ghost images. Here, we describe novel strategies to further improve the performance of isoSTED nanoscopy by reshaping the PSF. In particular, we propose employing moderate defocus on the depletion beam to reduce the side minima. Furthermore, we demonstrate a simplified alternative based on objective misalignment and quantitatively compare the expected performance between the two approaches.