Imaging was performed using a custom-built upright 2P-STED microscope as previously described (see [10 (link),19 (link),23 (link)] for a complete description). In all the following, 2D-STED and z-STED refer to images acquired using pure donut or pure bottle beams, respectively. Gold bead samples were used to assess the donut or bottle beam quality, while optical resolution was assessed by imaging fluorescent beads immobilized on a glass coverslip. Samples were prepared with either gold beads (150 nm Gold nanospheres, Sigma-Aldrich) or fluorescent beads (yellow-green fluorescent beads, 170 nm in diameter, Invitrogen) placed on polylysine-coated #1 coverslips. Samples were left to dry overnight and then transferred to a microscope glass slide, where they were sealed with nail polish. In order to evaluate the effect of the coverslip tilt angle, samples were placed on a tiltable mechanical stage (TTR001/M, Thorlabs), enabling us to vary the tilt angle relative to the microscope objective axis in a precise and systematic way. STED PSFs were originally checked and optimized at a tilt angle of 0 ± 0.03°.