Extended Data Fig. 1: Tissue-optimized STED.

a, b, STED light intensity distributions in the focal region, measured via backscattering from a 150 nm diameter gold nanosphere. a, Lateral (top) and axial (bottom) sections for 2π- and 4π-helical phase modulation. Scale bars: 250 nm. b, Axial sections of the π-top-hat phase modulated z-STED pattern (left), an incoherent superposition of 2π(xy)- and z-STED patterns (middle) and of 4π(xy)- and z-STED patterns (right). Power distribution between the z- and xy-STED patterns in the superpositions was 80% vs. 20%. Scale bars: 250 nm. c, Axial scan of 40 nm diameter Crimson fluorescent beads (Abberior) in confocal mode (left image) and with STED employing combined 4π(xy)- and z-STED patterns (right image) with the same power distribution as before. Scale bar: 250 nm. Profiles (2 pixel width) along the lines in lateral and axial directions as indicated in the image. Images are representative of n = 2 experiments. d, Extracellularly labeled neuropil in organotypic hippocampal slices. Orthogonal planes in xy- and xz-direction for diffraction-limited confocal (left), classical 2π-helical and π-top-hat phase modulation (middle), and combination of 4π-helical plus π-top-hat modulation for isotropic resolution with improved quenching of excitation outside the central STED intensity minimum (right). The 4π-helical pattern also facilitated robust in-tissue co-alignment of intensity minima. The images are representative of n = 3 technical replicates in two samples. Scale bars: 2 µm. Raw data with linear, inverted color scale. Numbers in greyscale bars refer to raw photon counts.