Stimulated emission depletion (STED) nanoscopy is one of a suite of modern optical microscopy techniques capable of bypassing the conventional diffraction limit in fluorescent imaging. STED makes use of a spiral phase mask to enable 2D super-resolution imaging whereas to achieve full volumetric 3D super-resolution an additional bottle-beam phase mask must be applied. The resolution achieved in biological samples 10 µm or thicker is limited by aberrations induced mainly by scattering due to refractive index heterogeneity in the sample. These aberrations impact the fidelity of both types of phase mask, and have limited the application of STED to thicker biological systems. Here we apply an automated adaptive optics solution to correct the performance of both STED masks, enhancing robustness and expanding the capabilities of this nanoscopic technique. Corroboration in terms of successful high-quality imaging of the full volume of a 15µm mitotic spindle with resolution of 50nm x 50nm x 150nm achieved in all three dimensions is presented.
The People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7/2007-2013) under REA grant agreement No 608133. The microscope development was supported by the Wolfson Foundation and the Scottish Universities Physics Alliance (SUPA). P. Z. would like to acknowledge that preparation of the paper was supported by National Science Center, Poland, under the project OPUS 13 (UMO- 2017/25/B/ST7/02049); Statutory funds of Faculty of Mechatronics of Warsaw University of Technology.
We would like to acknowledge the help of Michael Porter and Dr Iain Porter for the guidance and assistance in showing cell culturing, fixing and immunostaining protocols. We would also like to thank for the help of Dr Brian Patton for his useful tips regarding the optical setup. P. Z. would like to acknowledge Dr Maciej Trusiak for fruitful discussion and comments.