2000-fold parallelized dual-color STED fluorescence nanoscopy.

Stimulated Emission Depletion (STED) nanoscopy enables multi-color fluorescence imaging at the nanometer scale. Its typical single-point scanning implementation can lead to long acquisition times. In order to unleash the full spatiotemporal resolution potential of STED nanoscopy, parallelized scanning is mandatory. Here we present a dual-color STED nanoscope utilizing two orthogonally crossed standing light waves as a fluorescence switch-off pattern, and providing a resolving power down to 30 nm. We demonstrate the imaging capabilities in a biological context for immunostained vimentin fibers in a circular field of view of 20 µm diameter at 2000-fold parallelization (i.e. 2000 “intensity minima”). The technical feasibility of massively parallelizing STED without significant compromises in resolution heralds video-rate STED nanoscopy of large fields of view, pending the availability of suitable high-speed detectors