Stable stimulated emission depletion imaging of extended sample regions

Abstract

Stimulated emission depletion (STED) nanoscopy has become one of the most used nanoscopy techniques over the last decade. However, most recordings are done in specimen regions no larger than 10-30 × 10-30 μm2 due to aberrations, instability and manual mechanical stages. Here, we demonstrate automated 2D and 3D STED nanoscopy of extended sample regions up to 0.5 × 0.5 mm2 by using a scanning system that maintains stationary beams in the back focal plane. The setup allows up to 80-100 × 80-100 μm2 field of view (FOV) with uniform spatial resolution, a mechanical stage allowing sequential tiling to record larger sample areas, and a feedback system keeping the sample in focus at all times. Taken together, this allows automated recording of theoretically unlimited-sized sample areas and volumes, without compromising the achievable spatial resolution and image quality.