STED microscope with spiral phase contrast.

ABSTRACT

Stimulated Emission Depletion (STED) microscopy enables superresolution imaging of fluorescently marked nano-structures in vivo. Biological investigations are often hindered by the difficulty of relating super-resolved structures to other non-labeled features. Here we demonstrate that the similarity in optical design of Spiral Phase Contrast (SPC) and STED microscopes allows straightforward implementation of a phase contrast channel into a STED microscope in widefield and scanning modes. This method allows dual imaging and overlay in two contrast modes in fixed and in living specimens, in which double labeling is especially challenging. Living GFP- and YPF-stained neurons are imaged in one label-free phase contrast and one high-resolution STED channel. Furthermore, we implement SPC in widefield and scanning modes demonstrating that scanning confocal SPC yields the highest optical contrast. The latter configuration can provide contour detection or highlights and shadows reminiscent of differential interference contrast.