Gao, P. et al. Nat. Photonics 11, 163–169 (2017).

In stimulated emission depletion (STED) microscopy, a powerful super-resolution microscopy approach, an excitation laser and a donut-shaped STED laser are scanned together over a field of view. The excitation laser excites all fluorophores in an area, and the STED laser sends fluorophores into a dark state, leaving only the center of the donut fluorescent. Gao et al. have developed double-depletion STED, or STEDD. In STEDD, artificial background intensity is removed by the addition of a second STED pulse to conventional STED; this second, delayed pulse specifically depletes the fluorescent center of the donut, leaving only background. By using time-resolved detection, the researchers removed background, thus improving their super-resolved images. They demonstrated improved resolution when imaging fluorescent beads as well as labeled microtubules in mammalian cells.